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Phytophthora and Oak Decline - Impact on Seedlings and Mature Trees in Forest Soils

Jönsson Belyazid, Ulrika

2004

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Citation for published version (APA):Jönsson Belyazid, U. (2004). Phytophthora and Oak Decline - Impact on Seedlings and Mature Trees in ForestSoils. Ulrika Jönsson, Department of Ecology (Plant Ecology and Systematics), Ecology Building, LundUniversity, 223 62 Lund,.

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PHYTOPHTHORA AND OAK DECLINE

– IMPACT ON SEEDLINGS AND MATURE TREES IN FOREST SOILS

Phytophthora and Oak Decline– Impact on Seedlings and Mature Trees in Forest Soils

Ulrika Jönsson

ACADEMIC DISSERTATIONfor the degree of Doctor of Philosophy in Plant Ecology, to be publicly defended on November

19th 2004, at 10.00 a.m. in Blå Hallen at the Department of Ecology, Ecology Building,Sölvegatan 37, Lund, by permission of the Faculty of Natural Sciences of the University of Lund.

The thesis will be defended in English.

Faculty opponent: Professor Malcolm Press, Department of Animal and Plant Sciences,University of Sheffield, UK.

DissertationLund 2004

Till Mamma och Pappa

A doctoral thesis at a university in Sweden is produced either as a monograph or as a collection of papers. In thelatter case, the introductory part constitutes the formal thesis, which summarizes the accompanying papers.These have either already been published or are manuscripts at various stages (in press, submitted or in ms).

Layout: Gunilla Andersson/ZooBoTech & GrafikGruppenProofreading: Ulrika Jönsson

Printed by Xanto Grafiska AB, S Sandby

ISBN 91-7105-214-3SE-LUNBDS/NBBE-04/1076+126 pp

Cover: photo by Ulrika Jönsson

Phytophthora and Oak Decline – Impact onSeedlings and Mature Trees in Forest Soils

AbstractThis thesis investigated the occurrence of soilborne species of the plant pathogenic genus Phytoph-thora in southern Swedish oak forests and their possible involvement in southern Swedish oak de-cline. The emphasis was on the impact of P. quercina on Quercus robur in acid forest soils. Severaldifferent methods, including screening for Phytophthora in oak forest soils, greenhouse experimentswith seedlings grown in soils inoculated with Phytophthora, and field studies of mature oaks growingin Phytophthora-infested stands, were used. The results showed that Phytophthora species occur insouthern Swedish oak forests. The most frequently recovered species was P. quercina, which wasfound in 10 of 32 investigated oak stands. In addition, P. cactorum and P. cambivora (mating typeA2) were recovered from one stand each. The soil conditions at the sites from which Phytophthoraswere recovered ranged from mesic sediments to moraines, with clayey to silty textures and withpH(BaCl2) in the rhizosphere soil between 3.5 and 5.0.

In greenhouse studies, P. quercina and P. cactoum were found to infect and cause substantialdamage to roots of Q. robur seedlings grown in acid forest soils under a mesic water regime. Thepresence of the natural soil microflora did not hinder the pathogens from infecting and damagingthe roots. In addition, significant differences in live fine-root length were found between healthymature oaks and declining mature oaks growing in stands infested with P. quercina. No such differ-ence was found between trees growing in non-infested stands. The impact of the pathogen onmature oaks seemed to depend on tree vitality, site and climatic conditions. Despite the significantreductions in live fine-root length of both seedlings and mature oaks, few differences in above-ground growth (measured only in seedlings) and leaf nutrient concentrations were detected. How-ever, it seems likely that continuous root infections, with subsequent replacement of roots, willdeplete the carbohydrate stores in the plant and cause reductions in the production of new root andshoot tissue as well as in the production of secondary metabolites. This may result in an increasedsusceptibility of the tree to further pathogen infections as well as to other stress factors. A weakassociation was found between the occurrence of P. quercina and the vitality of oak stands (deter-mined from estimates of crown defoliation).

The impact of soil type on carbon allocation patterns in plants and its consequences for theextent of root damage caused by P. quercina is discussed, as well as the influence of abiotic and bioticfactors on the aggressiveness of Phytophthora, susceptibility of oak and subsequent disease develop-ment.

This thesis is based on the following papers, which are referred to by their Romannumerals.

I Jönsson U, Lundberg L, Sonesson K and Jung T. 2003. First records 47of soilborne Phytophthora species in Swedish oak forests. – Forest Pathology33: 175–179.

II Jönsson U, Jung T, Rosengren U, Nihlgård B and Sonesson K. 2003. 55Pathogenicity of Swedish isolates of Phytophthora quercina to Quercusrobur in two different soils. – New Phytologist 158: 355–364.

III Jönsson U. 2004. Phytophthora species and oak decline – can a weak 67competitor cause significant root damage in a nonsterilized acidic forestsoil. – New Phytologist 162: 211–222.

IV Jönsson U and Rosengren U. Can Phytophthora quercina have a negative 81impact on mature pendunculate oaks under field conditions? – Manuscript.

V Jönsson U, Jung T, Sonesson K and Rosengren U. Relationships 105between Q. robur health, occurrence of Phytophthora species and siteconditions in southern Sweden. – Submitted manuscript.

Papers I–III are reprinted with permission from the publisher.

Contents

Preface 11

1. Background 111.1 Oak decline – symptoms and causal agents 11

1.1.1 Biotic factors 121.1.2 Abiotic factors 121.1.3 Interactions between biotic and abiotic factors 13

1.2 Oak decline in southern Sweden 14

2. Aims and Hypotheses 14

3. Methods 153.1 Detection of Phytophthora species 163.2 Inoculation method for pot experiments with seedlings 163.3 Excavation of roots in the field 163.4 Determining the effects of Phytophthora spp. on the root systems 16

4. Phytophthora and Oak Decline 174.1 An introduction to the genus Phytophthora 174.2 Occurrence of Phytophthora species in European oak ecosystems and their 18

involvement in European oak decline4.3 Occurrence of Phytophthora species in oak forests in southern Sweden 194.4 Infection and symptoms in oak roots 194.5 Quantitative effects of Phytophthora on oaks in natural forest soils 20

4.5.1 Effects on root systems of seedlings grown in forest soil under controlled conditions 214.5.2 Effects on root systems of mature trees 224.5.3 Effects on crown vitality, above-ground growth and mineral nutrition 23

5. Influence of Abiotic and Biotic Factors on the Host – Pathogen Relationship 235.1 Abiotic factors 24

5.1.1 Water availability 245.1.2 Soil chemical properties 24

5.2 Biotic factors 26

6. Soil Type, Carbon Allocation and the Extent of Root Damage 27

7. A Conceptual Model for the Disease Development in Oak 30

8. Oak Decline in Southern Sweden – A Hypothesis 32

9. Conclusions 35

10. Future Perspectives 36

Acknowledgements 36

References 37

Thank you 43

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PrefaceToday, most scientists consider European oakdecline to be a complex phenomenon in whichdifferent site-specific factors interact with a suc-cession of abiotic and biotic factors causing amulti-faceted tree decline. My research has fo-cused on one part of this complex of factors,namely the plant pathogens Phytophthora, andthe primary aim of the work was to evaluate theinvolvement of soilborne Phytophthora species,particularly P. quercina, in southern Swedishoak decline. As a plant ecologist, I have tried toapproach the subject of plant pathology froman ecological point of view, evaluating the im-pact of P. quercina on its host, Quercus robur,under more natural conditions. This approachwas chosen since studies where maximum infec-tion and disease development are induced areabundant in the literature, while attempts tosimulate more natural scenarios are largely lack-ing.

I once read in the proceedings from a pathol-ogy conference on Phytophthora (Phytophthorain Forests and Natural Ecosystems, 2nd Interna-tional IUFRO Working Party Meeting in Alba-ny, W. Australia, 2001) that the subject of PlantEcology is “The quantification of the obvious”.However, I hope I have managed to bring someecological knowledge to the involvement ofPhytophthora species in European oak declineand especially, to their involvement in southernSwedish oak decline.

1. Background

1.1 Oak decline – symptoms and causalagents

Several studies performed during the past twodecades have shown extensive decline of Quercusspecies throughout Europe and North America,indicating episodes of severe stress followed byhigh mortality (Tainter et al., 1983; Oleksyn &Pryzbyl, 1987; Hartmann et al., 1989; Ragazziet al., 1989; Anonymous, 2000; Oszako, 2000;Rizzo et al., 2002). In Europe, pedunculate oak(Quercus robur) is the species which has shownthe most marked crown defoliation (Anony-mous, 2000). According to the UN/ECE Fed-eral Research Center for Forestry and ForestProducts, 35% of the pedunculate oaks in Eu-rope had a crown defoliation that exceeded25% in 1999 (i.e. could be considered dam-aged; Anonymous, 2000). Primarily two differ-ent types of decline have been identified; a rapidmortality where apparently healthy trees diewithin one or two growing seasons, and, a slow-er chronic decline, where trees gradually weak-en before they die, or, alternatively, recover. Thetwo types of decline are characterized by a varie-ty of symptoms exhibited by the oak trees, in-cluding thinning of the crown, formation ofleaf clusters, die-back of branches, discolorationor yellowing of the leaves, formation of epicor-mic shoots, exudations from the bark of thelower trunks, and bark lesions, many of which

Phytophthora and Oak Decline – Impact onSeedlings and Mature Trees in Forest Soils

12

are nonspecific and may be due to several differ-ent causal agents. Despite the low specificity ofthe symptoms and the different patterns of dis-ease development, the search for a commoncause behind oak decline, especially across Eu-rope, has been intense, and many different fac-tors have been suggested to trigger the outbreakof the damage (Führer, 1998; Oszako, 2000;Thomas et al., 2002).

1.1.1 Biotic factorsAmong the biotic factors, defoliation by insectlarvae during several consecutive years seems tobe the most accepted explanation of the onset ofoak decline. All over Europe (for example, inFrance, Germany, Hungary, Poland, Romaniaand Russia), various investigations have provid-ed evidence that defoliation by larvae of phyllo-phagous insects plays a dominant role in theoutbreak of oak decline. The most importantdefoliating insects seem to be Operophthera bru-mata L., Tortrix viridana L. and, in warmer re-gions, Lymantria dispar L. (Thomas et al.,2002). Bark beetles, borers and pathogenicmicroorganisms, on the other hand, have most-ly been regarded as secondary agents, killingonly already severely weakened trees (Manion,1991; Wargo, 1996). However, the extensivenecrotic areas and the increased tylosis of stemsand roots of declining oak trees, indicating in-fection by biotic agents (Luisi et al., 1993; Blas-chke, 1994; Kaus et al., 1996; Jung, 1998), di-rected some interest to the involvement of path-ogenic microorganisms in European oak de-cline. In the literature, it has been suggested thatseveral different pathogens, for example, Armil-laria species, Collybia fusipes, Fusarium eumar-tii, Microsphaera alphitoides, mycoplasma-likeorganisms, bacteria and viruses, are involved inthe decline process (Nienhaus, 1987; Ragazzi etal., 1993; Ahrens & Seemüller, 1994; Schlag,1995; Wargo, 1996; Marcais et al., 1999; Piouet al., 2002: Biosca et al., 2003; Camy et al.,2003). Most attention has been directed to-wards a group of Oomycota, Phytophthora spe-cies, almost all of which are aggressive primaryplant pathogens (Brasier et al., 1993; Jung &Blaschke, 1996; Jung et al., 1996; 1999; 2000;Robin et al., 1998; Vettraino et al., 2002; Balci& Halmschlager, 2003a,b; see also Chapter 4).

If attacks are repeated, borers, especially Agrilusbiguttatus Fabr. (Buprestidae), may also causegirdling and death of initially healthy trees(Hartmann, 1996).

1.1.2. Abiotic factorsOf the abiotic factors, three main groups havebeen considered to contribute to oak decline:climatic extremes, site conditions and air pol-lutants. Climatic extremes and site conditionsthat are unfavourable for the growth of Quercushave been widely recognized to significantly af-fect the health of the trees. Of the climatic fac-tors, changes in precipitation patterns and sum-mer droughts seem to be frequently associatedwith aggravated decline (Oosterbaan & Nabu-urs, 1991; Gibbs & Greig, 1997; Siwecki &Ulfnarski, 1998; Thomas & Hartmann, 1998),but exceptionally cold winters have also beenfound to have major damaging effects on oaktrees, with large areas of bark being killed (Hart-mann et al., 1989; Hartmann & Blank, 1992;Barklund & Wahlström, 1998; Barklund,2002). Spring frost is believed to occasionallycontribute to oak decline (Hartmann & Blank,1998). With regard to site conditions, close cor-relations were found between soil water condi-tions and tree health. In several central Europe-an countries, oak decline was found to be in-creased at hydromorphic sites with fluctuatingwater tables (Thomas et al., 2002). Air pollut-ants, on the other hand, are generally not be-lieved to contribute to oak decline. Thomas etal. (2002) concluded in their review of oak de-cline in central Europe that there is no evidenceof substantial contributions by any gaseous airpollutant (SO2, ozone, gaseous N compounds)to recent outbreaks of oak decline, and the stud-ies performed by Berger & Glatzel (1994), Tho-mas & Kiehne (1995), Simon & Wild (1998)and Thomas & Büttner (1998) on leaf and soilnutrient concentrations in oak stands supportthe suggestion that air-pollutant-inducedchanges in the nutritional status of the trees isnot a causal factor behind oak decline, at leastnot in Austria or Germany. Although few directcorrelations between nutrient status and oakhealth have been found, chemical changes insoil and trees resulting from a high input of Nand acidifying compounds may destabilize oak

Phytophthora and Oak Decline

13

ecosystems and thereby reduce the stress toler-ance of trees. Excess N may, under certain cir-cumstances, reduce the frost hardiness of oaks(Thomas & Blank, 1996; Thomas & Ahlers,1999), increase the susceptibility of trees to par-asite attack (Flückiger & Braun, 1998) or, incombination with drought, possibly lead to re-duced concentrations of allelochemicals in theleaves of Q. robur (Thomas & Schafellner,1999; Thomas et al., 2002).

1.1.3 Interactions between biotic and abioticfactorsAlthough many and diverse, the various hy-potheses presented above have usually ex-

plained only regional and short-term incidentsof oak decline. No general long-term, large-scale factor has yet been found. These findings,together with the low specificity of the symp-toms of decline, have led many scientists to be-lieve that the decline of oak is a complex phe-nomenon where several different abiotic andbiotic factors interact according to the spiraltheory for tree decline suggested by Manion(1991). Manion divided the causal factors intothree groups: predisposing, inciting and con-tributing (Figure 1). The predisposing factorsdecrease the vitality of the trees over a long pe-riod of time, thereby increasing their sensitivityto the inciting and the contributing factors.

Figure 1. Disease spiral for tree decline showing predisposing, inciting and contributing factors suggested tocontribute to oak decline (modified after Manion, 1991). The predisposing factors decrease the vitality of thetree over a long period of time, thereby increasing its susceptibility to inciting and contributing factors. Theinciting factors act over a shorter time and cause direct damage to tree tissue, while the contributing factors actonly on severely weakened trees.


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The inciting factors act on a shorter time scaleand cause direct damage to the tree tissue. Thecontributing factors act on the tree when it isalready severely weakened and usually haveconsiderable effects on the mortality level ofthe stand. The contributing factors are often ofbiotic origin. With regard to oak decline, thefactors suggested to be involved fall into allthree of these groups (Figure 1). In addition tothe factors already mentioned, forest manage-ment practices attempting to achieve high pro-duction on soils not suitable for oak produc-tion are likely to influence disease severity.Which of the factors in Figure 1 has the great-est impact on the decline of oaks, and howthese different factors interact with each other,subsequently causing a complex course of de-cline, is still unclear and requires further inves-tigation. However, Thomas et al. (2002) sug-gested a conceptual model for northern Ger-many involving three of these factors: insectdefoliation, summer drought and winter/spring frosts. Normally, at least two of thesefactors (although always including defoliation)have to occur simultaneously to trigger decline.These three factors are regarded as decisivecausal factors for past, as well as present, occur-rences of oak decline. However, single factorsare likely to be responsible for degradation andtree die-back in specific stands.

1.2 Oak decline in southern Sweden

Similar to the situation in the rest of Europe,oaks in southern Sweden (primarily Q. robur),have shown a dramatic deterioration in healthduring recent decades. In 1999, the proportionof damaged trees (crown defoliation > 25%) inthe southern part of the country reached a levelof 59%, compared with 9% in 1988 (Sonesson& Anderson, 2001). Barklund & Wahlström(1998) suggested that the initial cause of thedecline was severe bark necrosis, due to frostdamage caused by the unusually cold winters atthe end of the 1980s. However, very little re-search has been done in Sweden to elucidateother possible causes of the decline. A study wastherefore initiated to investigate whether Phy-tophthora species, which had previously been

suggested to be involved in central Europeanoak decline, were present in southern Swedishoak forests and, if they were, whether theymight be involved in oak decline in southernSweden.

2. Aims and HypothesesThe primary aim of this work was to evaluatewhether soilborne Phytophthora species werepresent in southern Swedish oak forests, and ifthese pathogens might be involved in southernSwedish oak decline (Papers I–V). In addition, Iwanted to evaluate at what kind of sites Phy-tophthora species occur (Paper V), and how dif-ferent abiotic and biotic environments affectthe development of disease in the oaks (PapersII, III & IV). In particular, I was interested instudying the influence of soil type (Paper II)and microbial competition (Paper III) on thepathogenicity of P. quercina to Q. robur. Basedon this, the following hypotheses were tested.(i) Phytophthora species are present in south-

ern Swedish oak stands. Soils in whichthese pathogens occur are less acidic andmore nutrient-rich than soils without thepathogen (Papers I & V)

(ii) Swedish isolates of Phytophthora can infectroots and cause damage to root systems ofseedlings grown in acid forest soils in thegreenhouse. However, the effects of thepathogens will be less severe in an acid for-est soil than in a nutrient-rich soil with ahigher pH (Papers II & III).

(iii) P. quercina can infect, and cause damage, toroot systems of mature oaks under fieldconditions (Paper IV).

(iv) Above-ground growth and nutrient con-centrations in leaves are negatively affectedby the presence of P. quercina in the soil(Papers II, III & IV).

(v) Phytophthora species are more likely to oc-cur in declining than in healthy stands (Pa-per V).

In addition, the importance of soil chemistryand climate for oak decline at 32 sites in south-ern Sweden was briefly evaluated (Paper V). Aschematic presentation of the factors that havebeen studied in this work are shown in Figure 2.


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3. MethodsSeveral different approaches were used to inves-tigate the involvement of Phytophthora speciesin southern Swedish oak decline. In the firststudy (Paper I), rhizosphere soil from 32 oakstands was sampled in order to determinewhether Phytophthora species were present at allin southern Swedish oak stands. A combination

of two greenhouse experiments, where oakseedlings (Q. robur L.) were grown under con-trolled conditions (Papers II & III), and a fieldexperiment using mature oaks (Paper IV), wasthen used to evaluate the aggressiveness of Phy-tophthora to the root systems of Q. robur. In ad-dition, the association between the presence ofPhytophthora and the state of decline of the


Figure 2. A schematic presentation of the factors studied within the scope of this thesis.

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stand was determined (Paper V). All the meth-ods used are described in Papers I–V. Some ad-ditional comments on the methods used andfeatures of specific interest are given below.

3.1 Detection of Phytophthora species

The soil samples used for detection of Phytoph-thora species were analysed using the soil baitingmethod described by Jung et al. (1996; 2000;Figure 3). This method was proven to be themost efficient for the detection of Phytophthoraspecies that occur in oak forest soils comparedwith several other diagnostic methods (Jung etal., 1996; Jung, 1998). Since Phytophthora spe-cies are slow growing, their presence is oftenmasked on agar plates by faster growing Py-thium species, which are commonly found insouthern Swedish oak forest soils (Paper I). Tominimize contamination by Pythium species,soil was dried at room temperature (17–20°C)before baiting (Jung et al., 1996), and slight al-terations in the composition of the selectivePARPNH agar described in Paper I was made.Soil drying for one week or more proved mostuseful in reducing the growth of Pythium spp.and other fast-growing fungi.

3.2 Inoculation method for pot experimentswith seedlings

In both greenhouse experiments, the inoculaand soil were mixed thoroughly before the seed-lings were transplanted (Papers II & III). Thisensured a more uniform distribution of Phy-tophthora propagules in the soil. This approachseemed most appropriate considering that a re-stricted flooding regime was applied and thatthe experiments were run for a relatively shorttime. If extensive flooding had been applied, orif water availability had been unlimited, theability of Phytophthora zoospores to disperse inthe entire soil volume by themselves would havebeen greater, and the method of inoculationwould have been less important for the develop-ment of disease (Robin et al., 2001). Theamount of primary inoculum used in the path-ogenicity tests has been used previously in a

number of studies (Jung et al., 1996; 1999;2003a,b). The amount of inoculum is likely tobe less important for the extent of damagecaused by the pathogens, due to the multicyclicnature of Phytophthora diseases (Erwin & Ribei-ro, 1996), implying that inoculum begets inoc-ulum within a relatively short period of time(hours) and continues to do so as long as envi-ronmental conditions favour rapid reproduc-tion and dissemination of sporangia andzoospores.

3.3 Excavation of roots in the field

Excavation of monoliths was chosen as the mostappropriate method of collecting roots in thefield (Paper IV). Soil coring is difficult in thesesoils, since there are many stones and rocks.Monoliths also give a more coherent root sys-tem than cores. However, the disintegration ofthe root system when collecting, washing andscanning the roots was still found to be too ex-tensive for reliable measurements of thenumber of fine roots and the number of fine-root tips per unit length of mother root. Furtherinvestigations are therefore needed to determinepossible differences in the branching patternbetween infected and non-infected mature trees(and seedlings).

3.4 Determining the effects of Phytophthoraspecies on the root systems

Visible signs of infection on the roots, recoveryof Phytophthora from necrotic tissue, and meas-urements of root length and biomass were usedto determine the impact of Phytophthora specieson oak root systems (Papers II, III & IV). Signsof infection were determined by comparing thesymptoms in oak seedlings and mature treesgrown in infested soil with seedlings and treesgrown in soils without Phytophthora and withsymptoms described in the literature (Blaschke,1994; Jung, 1998; Jung et al., 1996; 1999).

To avoid uncertainties due to individual dif-ferences in the handling of roots, the same per-son washed all roots included in an experiment.Separation of roots into living and dead was al-


17

ways done by me. Since the surface area and thevolume of roots were highly correlated with theroot length, and the results of the statisticalanalysis were similar, only root length is dis-cussed in the papers. In addition to the meas-urements of length, area and volume of roots,their dry weight was determined. For fine roots(0–2 mm), root length was found to be a moresensitive parameter than biomass.

To investigate the occurrence of Phytophthoraspecies in southern Swedish oak stands, to deter-mine if these isolates are pathogenic to oak rootsystems in acid forest soils, and to evaluatewhether these pathogens are involved in south-ern Swedish oak decline, the methods describedin Papers I–V seem appropriate and comple-ment each other. However, more thorough in-vestigations in the laboratory, employing meth-ods developed to answer only one or a few spe-cific questions, are required to determine thespecific role of Phytophthora species in southernSwedish oak decline, and the interactions be-

tween environmental factors, aggressiveness ofP. quercina, host susceptibility, and subsequentdisease expression.

4. Phytophthora and Oak Decline4.1 An introduction to the genusPhytophthora

Phytophthora species are a genus of fungus-likemicroorganisms that belong to the phylumOomycota in the kingdom Chromista (Figure4). Species of Phytophthora cause a variety of dis-eases in many different types of plants, rangingfrom seedlings of annual crops to mature foresttrees. Most species cause root rot, damping offof seedlings, and rot of lower stems and tubers(mainly soilborne species). Others cause rot orblight of buds, fruit or foliage (mainly airbornespecies; Erwin & Ribeiro, 1996; Agrios, 1997).Soilborne Phytophthoras occur worldwide, andcause damage to their hosts in nearly every partof the world where the soil becomes too wet forgood growth and the temperature remains fairlylow (Erwin & Ribeiro, 1996; Agrios, 1997),since these conditions favour the reproductionof the pathogens as well as increase the suscepti-bility of the host. Unlike most other soilbornefungal pathogens, Phytophthoras attack onlyfresh healthy plant tissue or newly exposedwounds, but do not invade tissue previouslycolonized by other microorganisms. Phytoph-thora, therefore, is almost never a secondarypathogen (Tsao, 1990). In all hosts affected by


Figure 3. The soil baiting method used for detectionof Phytophthora species from soil samples (Photo T.Jung).

Figure 4. Sporangia and zoospores of P. quercina(Photo T. Jung).

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Phytophthora root rot, many of the small andfine roots are dead and necrotic brown lesionsare often present on the larger roots. Some Phy-tophthora species attack the stem, where theycause water soaked and dark lesions in the bark.The infected bark area usually spreads and may,under certain circumstances, encircle the entirestem, or if it is restricted to one side of the stem,become a depressed canker below the level ofsurrounding healthy bark. The cankers mayspread up into the trunk, and sometimes thebranches, or down into the root system. As thecankers spread and enlarge, they may girdle thetrunk or roots (Agrios, 1997). Phytophthora is acortical invader in the majority of cases, andgenerally does not extend deep into the wood orattack the xylem (Tsao, 1990).

Plants infected with root- and bark-rottingPhytophthora species first show symptoms ofdrought stress and starvation, and then becomeweakened and susceptible to attack by otherpathogens or abiotic factors. Annual plants andyoung seedlings of trees may be killed within afew weeks or months if environmental condi-tions are favourable for the pathogen (Agrios,1997). In older trees, the die-back of the rootsystem may be slower, and affected trees usuallydo not die until several years after initial rootinfection by Phytophthora (Tsao, 1990). Mean-while, above-ground symptoms such as sparsefoliage, shorter, cupped and yellow leaves anddie-back of twigs and branches may occur(Tsao, 1990; Agrios, 1997). In most cases whena large tree dies as a result of root rot, almost allthe fine roots will have succumbed to Phytoph-thora infection, and additional stress, such asadverse climatic conditions, is often present andaggravates the course of events (Tsao, 1990).Among the Phytophthora species causing severediseases in forest ecosystems, P. cinnamomi injarrah ecosystems (Eucalyptus marginata) inAustralia, P. lateralis on Port-Orford cedar in theUSA and P. alni sp. nov. on Alnus spp. in Europeare probably the most well-known (Shearer &Tippett, 1989; Brasier et al., 1995; Hansen,2003). Recently, the newly described airborne P.ramorum has caused widespread mortality ofcoast live oak (Q. agrifolia) and tan oak(Lithocarpus densiflorus) along the west coast ofUSA (Rizzo et al., 2002; Davidson et al., 2003).

4.2 Occurrence of Phytophthora species inEuropean oak ecosystems and theirinvolvement in European oak decline

During the past decade, it has been suggestedthat several different Phytophthora species areinvolved in the decline of oak in Europe. Brasieret al. (1993) suggested that P. cinnamomi con-tributed to the decline of Q. ilex and Q. suber inIberia. Blaschke (1994) observed progressivedeterioration of fine roots and mycorrhizal sys-tems in mature, declining Q. robur trees, andsuggested that the damage was caused by Phy-tophthora species. Since then, Phytophthora spe-cies have been recovered from oak stands grow-ing in a wide variety of soil conditions acrossEurope (Jung & Blaschke, 1996; Jung et al.,1996; 2000; Robin et al., 1998; Gallego et al.,1999; Hansen & Delatour, 1999; Vettraino etal., 2002; Balci & Halmschlager, 2003a). Incentral, western and southern Europe, a diversePhytophthora population is usually present inoak forests. Among the 13 Phytophthora speciesrecorded in Europe, P. quercina, P. citricola andP. cambivora are widespread, while others seemto be restricted to sites with a warmer climate (P.cinnamomi, P. cryptogea), wet sites (P. gonapody-ides, P. europea, P. uliginosa) or acid sites (P. pseu-dosyringae). Additionally, P. cactorum, P. megasp-erma, P. psychrophila and P. syringae are occa-sionally isolated (Brasier et al., 1993; Jung et al.,1996; 2000; 2002; 2003b; Robin et al., 1998;Hansen & Delatour, 1999; Bianco et al., 2000;Hartmann & Blank, 2002; Vettraino et al.,2002; Balci & Halmschlager, 2003a). Hitherto,Phytophthora species have been recovered fromrhizosphere soil and roots of Q. cerris, Q. frai-netto, Q. ilex, Q. palustris, Q. petraea, Q. pubes-cens, Q. robur, Q. rubra and Q. suber.

The mere presence of Phytophthora species ata site does not necessarily mean that they areinvolved in oak decline. However, several stud-ies have demonstrated a significant negative im-pact of these pathogens on oak fine-root sys-tems under controlled conditions (Jung et al.,1996; 1999; 2002; 2003a,b; Robin & Desprez-Loustau, 1998; Robin et al. 1998; Gallego et al.,1999; Sanchez et al., 2002; Papers II & III).Complementary field studies have led to thesuggestion that P. cinnamomi is involved in the


19

decline of Q. ilex and Q. suber in Spain, Portu-gal and France (Brasier et al., 1993; Robin et al.,1998; Sanchez et al., 2002), that several differ-ent Phytophthora species are involved in centralEuropean oak decline and oak decline in Turkey(Jung et al., 2000; Balci & Halmschlager,2003a,b), and that P. quercina is involved in oakdecline in Italy (Vettraino et al., 2002). Undercertain circumstances, Phytophthora species mayact as the single factor causing disease of oaks(Jung et al., 2000; Brasier et al., 1995), but thedisease process is usually influenced by variousabiotic factors, often extreme climatic condi-tions (Brasier et al., 1993; Erwin & Ribeiro,1996). Few studies have failed to find associa-tions between the presence of these pathogensand crown condition, but in France, no correla-tions could be detected between health of treesand the presence of Phytophthora (Robin et al.,1998; Hansen & Delatour, 1999; Camy et al.,2003). However, Hansen & Delatour (1999)stated that their sampling method was inade-quate for supporting firm conclusions aboutcorrelations between crown status and the Phy-tophthora population in the soil. Furthermore,Q. rubra has been shown to be relatively resist-ant to infection by Phytophththora (Marcais etal., 1996; Robin et al., 2001).

4.3 Occurrence of Phytophthora species inoak forests in southern Sweden

In contrast to the diverse population of Phy-tophthora species found in oak ecosystems in therest of Europe, only three Phytophthora specieswere found in soils of southern Swedish oakstands (Q. robur L.; Paper I). The oak-specificfine-root pathogen P. quercina was the most fre-quently recovered species. P. quercina seemed tobe geographically widespread in southern Swe-den and was recovered from 10 out of 32 sam-pled oak stands, ranging from the southernmostpart of Skåne to the northern part of the prov-ince of Kalmar. The findings of P. quercina insouthern Sweden extend the northern limit ofthis species and complement recent records,which extended the geographical range of thisspecies into Turkey in the south-east and theUK in the west (Balci & Halmschlager, 2003b;

Brasier & Jung, 2003). Besides P. quercina, P.cactorum and P. cambivora (mating type A2)were recovered from rhizosphere soil in onestand each. The limited number of Phytophtho-ra species found in the oak forests suggest thatthe climatic conditions or the predominantlyacid soils of southern Sweden may not be suita-ble for more than a few Phytophthora species.Another explanation may be that other Phy-tophthora species have not yet been introducedinto Sweden. However, since additional sam-pling (Paper IV) increased the frequency of iso-lation of P. quercina in the stands where it waspresent (compared with the findings presentedin Paper I), repeated sampling during differentseasons and investigations of a larger number ofsites are needed before any firm conclusions canbe drawn about the general presence or not ofother Phytophthora species in Swedish oak forests.

The soil conditions in the stands from whichPhytophthora species were recovered rangedfrom mesic sediments to moraines, with clayey,loamy and silty textures, and soil pH(BaCl2) inthe rhizosphere between 3.5 and 5.0 (Paper I).Oak stands with Phytophthora had a significant-ly higher pH(BaCl2) in the rhizosphere soil thanstands without Phytophthora (Paper V). Themost frequently occurring Phytophthora speciesin southern Sweden, P. quercina, seemed to oc-cur more frequently in clayey and loamy soilsthan in silty and sandy soils. In general, Phy-tophthora species were found at more nutrient-rich sites, primarily with regard to Ca, andwhere the proportion of base cations in relationto acid ions was high (Paper V). These resultsare consistent with findings in Germany (Junget al., 2000; Hartmann & Blank, 2002) and tosome extent with findings in Austria (Balci &Halmschlager, 2003a). Although the pathogensoccur mainly at relatively nutrient-rich siteswith a somewhat higher pH, it was shown byboth Jung et al. (2000) and in Paper I that Phy-tophthora species may occur in all but the mostacidic soils, i.e. soils with pH(BaCl2/CaCl2) <3.5.

4.4 Infection and symptoms in oak roots

Phytophthora species may attack the leaves (P.ramorum), stems and roots of oaks. Most com-


20

monly, fine roots and stems are infected, withsubsequent die-back of the fine-root system andnecroses and lesion development on the stemsand collars. The first description of symptomsin roots of declining oaks (Q. ilex and Q. suber)resembling those caused by Phytophthora infec-tion came from Spain and Portugal (Brasier etal., 1993). Brasier et al. (1993) described deadand dying structural roots up to 20 cm in diam-eter with substantial necrotic inner-bark lesions.Isolation attempts gave positive results for P.cinnamomi. In 1994, Blaschke found similarsymptoms in roots of Q. robur. He describedthe infection process as starting with limitednecrosis and maceration of the primary tissuesof fibrous roots. In advanced stages, the deterio-ration of non-woody roots progressed towardsthe suberized mother roots. Having spreadfrom shallow necrotic lesions in the peripheralphellem, some hyphal branches, when not im-peded in their growth by the formation of lig-nitubers in the innermost phellem cells, werecapable of colonizing secondary cortex tissue,and induced discoloration of the adjacent phlo-em. In advanced stages of necrosis, penetratinghyphae could also be found in phloem and xy-lem elements. In some cases, root rot extendedfrom non-woody fine roots into mother roots.Blaschke (1994) also observed tylose formationin the xylem of the oaks. Coralloid hyphae andthick-walled oospores, typical of Phytophthora,were present in the necrotic tissue (Jung, 1998).

Several investigations on oak seedlings, per-formed in artificial soil mixtures as well as innatural forest soils under controlled conditions,have verified the symptoms found by Brasier etal. (1993) and Blaschke (1994). For Q. robur,substantial die-back of fine roots in oak seed-lings infected with several different Phytophtho-ra species, including southern Swedish isolatesof P. quercina, have been demonstrated in anumber of studies (Jung et al., 1996; 1999;2002; 2003a,b; Jung, 1998; Papers II & III),and similar symptoms have also been observedin Q. ilex, Q. rubra and Q. suber infected with P.cinnamomi (Marcais et al., 1996; Robin & De-sprez-Loustau, 1998; Robin et al., 1998; Galle-go et al., 1999; Robin et al., 2001; Rodriguez-Molina et al., 2002). Apart from root rot of fineroots, P. quercina and the other Phytophthora

species may cause orange-brown discolorationat the site of invasion, necrotic lesions of differ-ing sizes and induce formation of abnormalbranching (Jung et al., 1996; Marcais et al.,1996; Robin et al., 1998; Rodriguez-Molina etal., 2002, Papers II & III). Furthermore, P.quercina has been shown to infect suberizedcoarse roots of Q. robur (Jung et al., 1996; Pa-pers II & III). Necrosis usually developed viainfection of non-suberized, lateral roots. Insome infected oak seedlings, Phytophthora spe-cies may cause death of the entire tap rot, andwhen environmental conditions are favourablefor the pathogen, the whole plant may succumbto the infection and die (Jung et al., 1996;1999; 2002; 2003b; Marcais et al., 1996; Galle-go et al., 1999; Rodriguez-Molina et al., 2002;Papers II & III). In stem inoculation tests, thepathogens were shown to induce oval to fusi-form, sometimes girdling, necrotic lesionswhich, in some cases, were associated with darkexudates (Jung et al., 1996; Luque et al., 2000;Brasier & Kirk, 2001). However, P. quercinawas only weakly pathogenic, or not pathogenicat all, in these tests (Jung et al., 1996; Balci &Halmschlager, 2003b; Bianco et al., 2003). P.quercina therefore seems to be aggressive only toroot systems of Quercus species. The symptomsof Phytophthora infection described by Brasier etal. (1993) and Blaschke (1994), and the symp-toms found in seedlings grown under control-led conditions, have also been observed in otherfield studies (Jung & Blaschke, 1996; Marcais etal., 1996; Jung, 1998; Robin et al., 1998; Junget al., 2000; Paper IV).

4.5 Quantitative effects of Phytophthora onoaks in natural forest soils

The aggressiveness of Phytophthora species isstrongly influenced by both environmentalconditions and the activity of other soil micro-organisms (Tsao, 1990; Erwin & Ribeiro,1996). Despite this, the soil infestation testsused to examine the pathogenicity of P. quercinaand other Phytophthora species have usuallybeen performed on oak seedlings grown in ster-ile mixtures of peat, vermiculite and sand, withrelatively high pH values and high concentra-


21

tions of mineral nutrients (Jung et al., 1996;1999; 2002; 2003a,b; Robin & Desprez-Lous-tau, 1998; Robin et al., 1998; 2001; Luque etal., 2000; Sanchez et al., 2002). Temperatureand water regimes applied in these pathogenici-ty tests are often designed to favour the patho-gen, in an attempt to maximize disease develop-ment. In these studies, the root rot is usually se-vere (often exceeding 50% of the root systemwhen aggressive pathogen isolates are used) andlarge lesions on roots may be formed, occasion-ally resulting in high mortality of the seedlings(Jung et al., 1996; 1999; 2002; 2003b; Robin& Desprez-Loustau, 1998; Robin et al., 1998;2001; Luque et al., 2000; Sanchez et al., 2002).Few studies have focused on the effects of thepathogens under more natural conditions, us-ing forest soils, which have an active soil micro-flora and also usually have higher acidity, loweramounts of nutrients, a lower water-holding ca-pacity and are more compacted than artificialpeat-vermiculite-sand mixtures. Exceptions aresome studies on the pathogenicity of P. cinnam-omi to Q. rubra and Q. ilex, where forest soilshave been used to a certain extent when assess-ing the interaction between the pathogen andthe host (Marcais et al., 1996; Gallego et al.,1999), and stem inoculation tests with Phytoph-thora isolates from Turkey on Q. petraea and Q.cerris grown in forest soils (Balci & Halmschlag-er, 2003b). Furthermore, few field studies, inwhich the quantitative effects of these patho-gens on mature trees in forests have been esti-mated, can be found in the literature (i.e. Junget al., 2000). Considering southern Swedish oakforests, it is particularly interesting to determinethe effects of Phytophthora species under morenatural conditions, since the majority of ouroak forest soils have pH(BaCl2) values below4.2, and it is common that the base saturation islower than 20% at 20–30 cm depth in the min-eral soil (Sonesson & Anderson, 2001). Theseare conditions known to be unfavourable toPhytophthora species (Schmitthenner & Canaday,1983; Erwin & Ribeiro, 1996; Jung et al., 2000).

4.5.1 Effects on root systems of seedlings grownin forest soil under controlled conditionsIn a first attempt to determine the ability of P.quercina to cause root damage in forest soils, the

pathogenicity of two southern Swedish isolatesof P. quercina to seedlings of Q. robur in anacidic, N-rich but otherwise nutrient-poor for-est soil was investigated (Paper II). In this exper-iment, the forest soil was autoclaved, to kill thenatural soil microflora. The experiment wastherefore limited to studying the pathogenicityof P. quercina in the chemical and physical envi-ronment of a forest soil. A restricted wateringregime and relatively high temperature, at-tempting to simulate southern Swedish sum-mer conditions, were applied. In contrast towhat was expected based on previous investiga-tions of the influence of soil chemical factors onPhytophthora diseases (Schmitthenner & Cana-day, 1983; Erwin & Ribeiro, 1996), the soil in-festation test showed that the physical andchemical environment of the forest soil did notinhibit P. quercina. The pathogen was recoveredfrom root fragments of all seedlings grown insoil infested with the pathogen, and was shownto cause significant root rot. Infected seedlingshad on average 34 to 35% of dead fine-rootlength compared with 15% in the control seed-lings. In addition, the dead root length of coars-er roots (2–5 mm in diameter) was significantlyhigher in infected seedlings, verifying the abilityof P. quercina to induce damage in suberized tis-sue. Results from this first experiment demon-strated that P. quercina can cause substantialdamage to the root systems of oak seedlings un-der, for the pathogen, adverse chemical andphysical conditions in the soil: low pH, highconcentration of Al, low concentration of Ca, acompact soil structure and restricted periods ofhigh water availability.

In the second experiment (Paper III), thesame forest soil, but not autoclaved, was used.The aim of this experiment was to investigatethe effects of P. quercina and P. cactorum in aforest soil when in competition with the naturalsoil microflora. This is necessary in defining therole of Phytophthora species in European oakdecline, since these pathogens are usually re-garded as weak competitors, and have been sug-gested to only infect plant tissue under condi-tions of reduced competition from rhizospheremicroorganisms due to the presence of water(Tsao, 1990, Erwin & Ribeiro, 1996). In thisexperiment, the seedlings were older (14–18


22

weeks old, compared with the 8–12-weeks oldseedlings in the first experiment), and the dura-tion of the experiment was longer (six monthsinstead of three months). The watering regimeand temperature were similar to those in thefirst experiment. The results showed that bothP. quercina and P. cactorum could cause fine-rootdecay, with die-back of non-suberized as well assuberized fine roots, despite the presence of thenatural soil microflora. Live fine-root lengthwas significantly higher in control seedlingsthan in seedlings infected with P. cactorum andall isolates of P. quercina, except one (seven iso-lates of P. quercina were tested). The reductionin the live fine-root length due to Phytophthorainfection varied between 31 and 64% depend-ing on the isolate. The proportion of dead rootlength in relation to total root length was onaverage 26%, while control seedlings had only7% dead root length. However, the length ofdead roots was influenced by the presence ofsaprophytic microorganisms, and thereby de-pended not only on the activity of Phytophtho-ra, but also on the decomposition rate. In ac-cordance with the first experiment, suberizedcoarser roots (diameter 2–5 mm) were also in-fected and the dead root length of these wassignificantly higher in infected seedlings thanin control seedlings. Obviously, P. quercina andP. cactorum can cause significant damage toroot systems of Q. robur seedlings also whenthe natural soil microflora is present in the for-est soil, in the absence of extended periods offlooding.

In contrast to previous studies, which haveoften attempted to maximize disease develop-ment by extended periods of waterlogging, theaim of these two greenhouse experiments was toexamine the pathogenicity of P. quercina (and toa lesser extent P. cactorum) to seedlings grownunder conditions designed to mimic a southernSwedish summer. The lower proportion of rootrot in these two studies, compared with severalprevious studies examining the pathogenicity ofP. quercina and other Phytophthora species toroot systems of oak seedlings, is therefore notsurprising, since the infection of fine roots byPhytophthora species and symptom expression isgenerally believed to be favoured by a highavailability of water (Erwin & Ribeiro, 1996).

However, it may also be due to chemical andphysical constraints of the soil on the pathogen.For further discussion of the effects of abioticfactors on the relationship between Phytophthoraand oak, see Section 5.1.

4.5.2 Effects on root systems of mature treesQualitative studies, determining the presence ofPhytophthora species at a site, their infection oftree tissue and their association with decliningoaks, are common in the literature (Brasier etal., 1993; Jung et al., 1996; 1999; 2000; Robin& Desprez Loustau, 1998; Robin et al., 1998;Gallego et al., 1999; Hansen & Delatour, 1999;Balci & Halmschlager, 2003a,b). However, lit-tle is still known about the quantitative effectson mature trees in oak forests (i.e. Jung et al.,2000). In southern Sweden, healthy, moderatelydeclining and severely declining trees in tenstands, five of which were infested with P.quercina and five which were not, were sampledto evaluate the impact of this pathogen on rootsystems of mature Q. robur trees (Paper IV).Each stand where the pathogen was present,was paired with another stand where the patho-gen was not present, in order to verify the im-pact of P. quercina. The matching of stands intopairs was primarily based on soil texture, soilchemistry and geographical location of thestands, but geological substrate, stand type andstand age were also taken into consideration.Results showed that live fine-root length perunit soil volume was significantly higher forhealthy than for declining trees in infestedstands. In non-infested stands, on the otherhand, there were no significant differences inlive fine-root length between trees. The impactof the pathogen was dependent on the season ofsampling, being most severe after an unusuallydry summer (August 2002), and least pro-nounced at the beginning of the growing season(June 2002). This suggests that there may be aninteraction between drought and Phytophthoraattack, supporting previous investigations onoak seedlings, where Jung et al. (2003a) demon-strated that P. quercina caused higher amountsof root damage to Q. robur under conditionswhere drought and flooding were alternatedthan when moist soil conditions prevailed be-tween flooding cycles.


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The data presented in Paper IV support theresults of a German study on the impact of Phy-tophthora species on mature trees of Q. robur,which also revealed significant differences in theroot systems between healthy and decliningtrees (Jung et al., 2000). In the German study,declining trees showed a higher amount of visi-ble root damage and significantly lower numberof fine roots per unit length mother root, lowerspecific root tip density and lower fine-rootlength per unit mother root length than healthytrees in 19 stands infested with Phytophthora(Jung et al., 2000). Similar to the Swedish study,there were few differences in root parametersbetween healthy and declining trees in the 16stands where the pathogen was not present.These results indicate that Phytophthora specieshave a significant impact on the live fine-rootlength, on the proportion of fine roots and fineroot tips and on the branching patterns of theroot systems of mature oaks under field condi-tions. In contrast to the Swedish study, almostall root parameters in the stands without Phy-tophthora in Germany were superior to those oftrees in stands with the pathogens. However,Jung et al. (2000) stated that these differencesmight be due to differences in site conditionsrather than to fine-root damage caused by Phy-tophthora. The influence of soil type on the ex-tent of damage caused by P. quercina on oakroot systems is discussed in Chapter 6.

4.5.3 Effects on crown vitality, above-groundgrowth and mineral nutritionSignificant associations between the presence ofPhytophthora, particularly P. quercina, in therhizosphere soil, fine-root damage (investigatedonly in Germany) and crown vitality of individ-ual oak trees have been found in several Europe-an countries (Jung et al., 2000; Vettraino et al.,2002; Balci & Halmschlager, 2003a,b). Insouthern Sweden, declining oaks growing instands infested with P. quercina were found tohave significantly lower live fine-root lengththan healthy trees (see Section 4.5.2; Paper IV).Furthermore, a weak association (p=0.088) wasfound between the occurrence of P. quercina insoil and the vitality of oak stands (determinedfrom estimates of crown defoliation), indicat-ing that oak stands were more likely to be de-

clining if P. quercina was present in the soil (Pa-per V).

Despite significant associations between thepresence of Phytophthora species, loss of fineroots and unhealthy appearance of the crown ofseedlings and trees, there are often none, or veryfew, effects on the above-ground growth andnutrient status of seedlings (Papers II & III;Wilcox et al., 1993; Jung et al., 1999; Sanchez etal., 2002) or on the nutrient status of maturetrees infected with Phytophthora (Paper IV).The small effects on seedlings suggest that thereis a significant timelag between root loss andabove-ground effects, or that the nutrient up-take efficiency of the remaining roots increaseswhen roots are lost due, for example, to patho-gen attack. Alternatively, plants may have anexcess of roots, which may be a trait that hasbeen of evolutionary advantage during periodsof multiple stress. Mature trees that are sufferingfrom fine-root losses usually have a reducedcrown and the fine-root system may thus beable to take up enough nutrients for the remain-ing crown. Nutrient deficiencies may thereforebe obvious only in severely declining trees. Re-sults from both the greenhouse experiments(Papers II & III) and the field experiment (Pa-per IV) are in good agreement with those of pre-vious studies, suggesting that Phytophthora mayinfect roots of woody plants several years beforefoliage symptoms are detected, and that root rotmust usually be severe before significant effectscan be seen on the aerial parts of plants (Tsao,1990; Maurel et al., 2001).

5. Influence of Abiotic and BioticFactors on the Host–PathogenRelationship

The development of diseases caused by Phy-tophthora is strongly influenced by a largenumber of abiotic and biotic factors and the lit-erature on the influence of these factors on Phy-tophthora epidemiology is too voluminous forcomplete review here. I have therefore empha-sized some factors that I believe are importantfor the development of Phytophthora diseases onoak in acid forest soils in a temperate climate.


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5.1 Abiotic factors

5.1.1 Water availabilityPhytophthora diseases are multicyclic, whichmeans that inoculum may amplify rapidlywhen the environmental conditions, the mostimportant of which is the presence of free water,are favourable (Erwin & Ribeiro, 1996). Theincrease in inoculum from low, often undetect-able levels to high levels within a few days orweeks, is caused by the rapid production of spo-rangia and zoospores at the surface of infectedplant tissues. The short regeneration time andgreat reproductive capacity imply that Phytoph-thora diseases may develop epidemically whenthe soil remains excessively wet for prolongedperiods and temperatures remain fairly low (Er-win & Ribeiro, 1996). However, consideringthe disease development of oak, drought or var-iations in water availability, especially fluctua-tions between drought and flooding, also seemto be detrimental to the fine-root systems (Bra-sier et al., 1993; Jung et al., 2003a). In particularP. quercina seems to be less dependent on soilmoisture for its survival under natural condi-tions (Hansen & Delatour, 1999; Jung et al.,2000; Balci & Halmschlager, 2003a), whichmay be due to its thick-walled oospores, mak-ing it well adapted to survive in dry soil condi-tions. In greenhouse experiments with P. querci-na and Q. robur, the pathogen was shown tocause substantial damage to root systems of oakseedlings under restricted mesic water regimes(attempting to simulate summer conditions insouthern Sweden; Papers II & III), demonstrat-ing that P. quercina can cause substantial dam-age not only when weather or site conditionsinvolve periods of high or unlimited water avail-ability. Similar results were also found for dis-ease development in Q. ilex and Q. suber infect-ed with P. cinnamomi (Robin et al., 2001;Sanchez et al., 2002). Furthermore, Jung et al.(2003a) demonstrated the ability of P. quercinato survive during periods of drought, and tocause higher amounts of root damage to seed-lings under conditions where drought andflooding were alternated than when moist soilconditions prevailed between flooding cycles.Drought also seemed to be a critical factor forthe degree of root damage caused by P. quercina

on root systems of mature trees in oak forests insouthern Sweden (Paper IV). Live root lengthper unit soil volume was significantly lower forboth moderately and severely declining oakscompared with healthy oaks after an unusuallydry summer (August 2002), while no differencecould be detected between healthy and moder-ately declining trees on the other sampling occa-sions (June 2002 and March 2003). The highamount of damage when Phytophthora anddrought interact may be due to the simultane-ous increase in the production of sporangiaupon rewetting and the production of new, un-suberized fine roots, which are highly suscepti-ble to infection. Extended periods of droughtmay also increase root exudation, thereby facili-tating the initial establishment of soilbornepathogens in the roots (Duniway, 1977), andmay, in the event of an increase in soil moisture,favour Phytophthora over other soil microorgan-isms, since Phytophthoras respond very rapidlyto changes in soil moisture (as can be seen whendrying the soil before soil baiting). Restrictedwater availability, or drought, may also criticallyreduce the tolerance of the host to the pathogenthrough its influence on the defence mecha-nisms of the plants. Horner (1990) suggestedthat severe drought may lead to a reduction instomatal conductance and, thus, carbon gain.These reductions can result in the preferentialallocation of C to primary metabolites and adecrease in the formation of secondary com-pounds, such as allelochemicals (Bryant et al.,1983). Severe drought may therefore increasethe risk of pathogen attack as well as defoliation.Performance of phyllophagous insects has beenshown to increase with reduced concentrationsof foliar allelochemicals (Joseph et al., 1993;Schafellner et al., 1994; Hättenschwiler &Schafellner, 1999). A mild water deficit, on theother hand, may lead to growth reduction priorto distinct reduction of stomatal conductance,thereby inducing an increase in C flow to sec-ondary metabolites and increased concentra-tions of secondary compounds (Kramer, 1983;Horner, 1990; Lambers et al., 1998).

5.1.2 Soil chemical propertiesMany different chemical properties of the soilhave been shown to influence the aggressive-


25

ness of Phytophthora species. In general, Phy-tophthora diseases are considered to be more se-vere at higher pH values (Schmitthenner &Canaday, 1983). For Phytophthora species oc-curring in oak stands, Jung et al. (2000)showed that sporangia can not be formed atpH(H2O) values below 4.0. In addition, theproduction of sporangia was found to increasewith increasing pH. Concentrations of Al andCa in the soil are often inversely and intimatelyrelated to soil pH, and may also affect the ag-gressiveness of Phytophthora. High concentra-tions of Al have been shown to inhibit mycelialgrowth, as well as sporangial formation andgermination in P. capsici and P. infestans (Mu-chovej et al., 1980; Andrivon, 1995; Erwin &Ribeiro, 1996). High concentrations of Camay suppress diseases caused by Phytophthoraspecies (Von Broembsen & Deacon, 1996;1997), but generally, the diseases are consid-ered to be more severe at high Ca levels (Sch-mitthenner & Canaday, 1983; Erwin & Ribei-ro, 1996). Minimum levels of Ca are necessaryfor zoospore production as well as cyst germi-nation (Halsall & Forrester, 1977; Von Broem-bsen & Deacon, 1996; 1997; Xu & Morris,1998), and Ca levels also affects Phytophthorainfection of roots through its effect on zoosporetaxis and adhesion to solid surfaces (Gubler etal., 1989; Deacon & Donaldson, 1993). Fur-thermore, external Ca stimulates oospore ger-mination (Ribeiro, 1983). As for Ca, high con-centrations of Mg usually increase the severityof disease caused by Phytophthora (Schmitthen-ner & Canaday, 1983). High concentrations ofK and P, on the other hand, are generallythought to decrease disease severity, or not af-fect it at all (Schmitthenner & Canaday, 1983;Graham, 1988; Rehm & Stienstra, 1993). Theinfluence of cations and P may depend on theconcentration of N and the ratio of the cationsand P to N, since application of complete ferti-lizer has been shown to decrease disease severity(Schmitthenner & Canaday, 1983; Pacumbabaet al., 1997).

The effect of N on disease development isvariable and seems to depend on both thehost–pathogen combination and the type ofsoil N (Klotz et al., 1958; Newhook & Podger,1972; Schmitthenner & Canaday, 1983;

Utkhede & Smith, 1995; Erwin & Ribeiro,1996). For P. quercina and Q. robur, Jung et al.(2003a) demonstrated a stimulation of in vitroproduction of sporangia with increasing con-centrations of nitrate in the soil leachate, andan increasing difference in fine-root length andthe number of fine-root tips between uninfect-ed and infected seedlings with increasing ni-trate concentration in the soil. In contrast tothis, the study described in Paper IV showedhigher concentrations of total N in the organicand uppermost mineral soil layers aroundhealthy and moderately declining mature treesin Phytophthora-infested stands, and these treesalso had higher N concentrations in the leavesthan severely declining trees. It seems likelythat under conditions of N limitation, the treesare favoured by good tissue N status, since it islikely to improve the trees’ ability to sustainroot production and replace roots destroyed bythe pathogen (see Chapter 6). It may also affectthe ability of the plants to produce defensivecompounds (Agrios, 1997; Crawley, 1997;Lambers et al., 1998). Studies of “little leafdisease” of shortleaf pine (Pinus echinata),caused by P. cinnamomi on degraded soils inthe south-eastern USA, have shown that fertili-zation with N may prevent symptom develop-ment of healthy trees and improve the condi-tion of little leaf trees in early stages of disease(Tainter & Baker, 1996). In situations of excessN, on the other hand, P. quercina is likely tohave an advantage, since smaller amounts ofcarbohydrates may be available in the tree forthe production of defensive compounds, andnutrient imbalances may render tissues moresusceptible to pathogen infection (Marschner,2003). A decrease in the protein precipitatingcapacity of trees and in the foliar concentra-tions of phenolics and tannins in response toincreased N has commonly been observed inboth gymnosperms and deciduous trees(Balsberg-Påhlsson, 1992; Muzika & Pregitzer,1992; Joseph et al., 1993; Schafellener et al.,1994; Hättenschwiler & Schafellner, 1999),and Thomas & Schafellner (1999) demon-strated that the combination of excess N anddrought led to a decline in foliar tannin con-centration and protein precipitation capacityof pedunculate oak seedlings.


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5.2 Biotic factors

A number of studies have shown that variousbiotic populations in the soil influence the se-verity of disease caused by Phytophthora species.Some organisms, such as nematodes, facilitatethe infection of Phytophthora by creatingwounds on the roots (Barham et al., 1974).Others accelerate the damage already caused byPhytophthora through secondary infections. Athird group reduces the amount of damagecaused by Phytophthora species through parasit-ism on the Phytophthora propagules in the soil(Erwin & Ribeiro, 1996), or by exerting com-petitive pressure on the Phytophthora species,thereby excluding them from the root surface(Weste & Vithanage, 1977; Marx, 1972; Keast& Tonkin, 1983; Malajczuk, 1983). In particu-lar, mycorrhizal colonization has been suggestedto protect roots against Phytophthora infection(Zak, 1964; Barham et al., 1974; Marx, 1972).

Zak (1964) suggested several differentmechanisms by which ectomycorrhiza mayprotect plants from diseases caused by rootpathogens. (i) The additional physical barrierformed by the mycorrhizal mantle may hinderzoospores and mycelia from penetrating roots.(ii) The utilization of surplus carbohydrates bythe mycorrhizal fungi may decrease the chemo-taxis of the pathogen to the root. (iii) The secre-tion of antibiotics by the fungus may reduce thepathogen inoculum potential. (iv) The ectomy-corrhizal fungi may, together with the root, sup-port a protective microbial rhizosphere popula-tion. Furthermore, Marx (1969) suggested thatthe symbiosis between the plant roots and thefungi might initiate changes in the host cellsthat inhibit infection and spread of the patho-gen. Mycorrhizal symbiosis may also enhanceplant nutrition, thereby reducing the host sus-ceptibility to pathogen infection (Linderman,1994). Several of these mechanisms probablyact synergistically, and their relative importanceprobably depends on the plant species involved,the specific ectomycorrhizal fungi and the pre-vailing environmental conditions.

The majority of studies aimed at investigat-ing the interactions between ectomycorrhizalfungi and Phytophthora species on woody plantshave been performed by Marx and colleagues

(Marx, 1969; 1970; 1973; Marx & Davey,1969a, b). Using agar plates and liquid cultures,Marx (1969) demonstrated an inhibitory effectof several ectomycorrhizal species on the growthof mycelia, as well as on the motility and germi-nation of zoospores of P. cinnamomi. Further-more, the presence of fully developed mycor-rhiza with a fungal mantle and a Hartig net onindividual roots of shortleaf pine seedlings wasseen to provide resistance to zoospore and myc-elial infection, and to have a protective influ-ence on adjacent non-mycorrhizal roots (Marx& Davey, 1969a,b; Marx, 1970). The protectiveeffect of the mycorrhizal symbiosis was evidentthroughout at the whole root system of seedlings(Marx, 1973). In contrast, non-mycorrhizalroots and roots with incomplete fungal mantleswere infected by the pathogen (Marx & Davey,1969a,b; Marx, 1970; Barham et al., 1974).Similar results were found by Branzanti et al.(1999) when investigating the importance ofectomycorrhizal fungi in the reduction ofchestnut ink disease. Mycorrhizal chestnutseedlings infected with P. cinnamomi or P. cam-bivora showed no signs of pathogen infectionand the growth of seedlings was not reduced bythe presence of the pathogens in the soil. Non-mycorrhizal seedlings, on the other hand, wereseverely affected by the infection of Phytophthora.

These results suggest that the presence ofother microorganisms in the rhizosphere soilmay render oak trees less susceptible to infec-tion by Phytophthora species, although root in-fection is most likely not completely inhibited(Paper III). In particular, ectomycorrhizal oakroots may be less affected by Phytophthora infec-tion than non-mycorrhizal ones, provided thatthe symbiotic relationship is well established.However, the influence of the soil microflora onPhytophthora species is likely to vary substantial-ly between sites and seasons, depending on thecomposition of the microbial community, themicrobial activity and the growth rate of thenew fine roots. Furthermore, low vitality of oaktrees (Causin et al., 1996; Kovacs et al., 2000)and a high input of N into ecosystems (Brun-ner, 2001; Nilsson, 2004), have been found toinfluence the ectomycorrhizal colonization ofroots negatively. Epidemiological studies undercontrolled conditions and field conditions are


27

thus required before the significance of ecto-mycorrhizal colonization of roots for disease re-sistance of oak can be determined.

6. Soil Type, Carbon Allocation andthe Extent of Root DamageThe negative influence of low pH and highconcentrations of Al on sporangial productionand mycelial growth and the necessity of mini-mum levels of Ca for zoospore production, cystgermination and the infection process (see Sec-tion 5.1.2), make it reasonable to assume thatPhytophthora species would cause less severedamage to root systems of oaks growing in acidforest soils than in the artificial soil mixtureswhich are commonly used in pathogenicitytests. Contrary to this, the study presented inPaper II showed that seedlings of Q. robur weremore severely affected by P. quercina whengrown in an acid, N-rich forest soil, than whengrown in a peat–sand mixture. This was ex-pressed as a greater reduction in live fine-rootlength, higher proportion of dead root lengthand infection of coarser suberized roots (PaperII). One possible explanation of these resultsmay be N-induced stimulation of sporangialproduction in the forest soil, promoting the in-fection of greater numbers of fine roots, therebydeteriorating the fine-root system, as was shownby Jung et al. (2003a) when enriching an artifi-cial soil with nitrate. However, since the growthof seedlings in the forest soil was reduced com-pared with the growth of seedlings in the peat–sand mixture (Figure 6a), I hypothesize that theresults are due to the influence of soil type onthe C fixation and C allocation patterns in theseedlings, thereby affecting the capacity of seed-lings to regenerate roots and produce defensivecompounds. It is well known that mineral nu-trients may affect root growth, morphology anddistribution of root systems in the soil (Lamberset al., 1998; Marschner, 2003). The effect isparticularly pronounced with N, but also P, S,K, Mg and Mn can affect the C allocation pat-terns in trees, thereby influencing the relationbetween root and shoot growth (Ericsson et al.,1996; Lambers et al., 1998, Marschner, 2003).Shortage of N, P and/or S usually results in an

increased allocation of C to the roots, therebyfavouring root growth over shoot growth, whilea shortage of K, Mg or Mn results in a decreasein the allocation of assimilates to the roots (Eric-sson et al., 1996; Marschner, 2003). To visualizea possible mechanism by which nutrient availa-bility may influence the C fixation and alloca-tion patterns within the tree, a causal loop dia-gram was constructed (Figure 5). The diagramis based on the theory for C allocation in treessuggested by Ericsson et al. (1996). The pur-pose of the causal loop diagram is to look at thebalance between root- and shoot-growth andallocation to secondary metabolites in the oakseedlings. The model thus only describes thesethree pools of carbohydrates in the tree and theflow of carbohydrates between them. It doesnot consider various groups of compounds orindividual compounds, and it does not explainthe physiological processes. In addition, I as-sume that the seedlings are not limited by water,light or CO2. The diagram is described below.

For seedlings grown in the acid N-rich forestsoil, K (or possibly Mg) is likely to limit growth,since the concentration of K in the soil and inthe leaves is low, especially when compared withN (Tables 1 & 2). Shortage of K, Mg, or Mn,elements that are important in photosynthesis(denoted photosynthesis nutrients in Figure 5),will result in a reduced rate of photosynthesisand subsequently, in reduced C fixation (Erics-son et al., 1996). This will impede the replen-ishment of the pool of non-structural carbohy-drates, and lower amounts of carbohydrates willthen be available for growth and the productionof secondary metabolites. Since the carbohy-drates are formed in the photosynthetically ac-tive part of the seedlings, the available carbohy-drates will probably be metabolized for growthin the above-ground part first (Ericsson et al.,1996), leading to low translocation of C to theroots and, consequently, low root growth com-pared with shoot growth. If root growth is de-creased, the uptake of base cations is likely to befurther impaired. In addition, the high availa-bility of N (or P, S) which is important to me-tabolize carbohydrates for growth (denotedgrowth nutrients in Figure 5; Ericsson et al.,1996; Marschner, 2003), may reduce theamount of carbohydrates allocated to secondary


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Figure 5. A causal loop diagram describing how soil type may influence the C allocation patterns in plants. CH= carbohydrates; photosynthesis nutrients = K, Mg, Mn; growth nutrients = N, P, S. Note that arrows onlyindicate the direction of the causality. An arrow with a positive causality sign implies that a change in theaffecting factor drives a change in the affected factor in the same direction. Arrows with negative causality signsimply a change in the opposite direction. For further information and a description of the diagram, see Chapter 6.

Table 2. Leaf nutrient concentrations1 of seedlingsgrown in an artificial peat–sand mixture and ofseedlings grown in an acid forest soil. Values givenare average values ± SD (mg g–1) for control seed-lings (n = 5).

Element Peat–sand Forestmixture soil

Ca 20.3 ± 3.2 12.5 ± 3.5K 13.6 ± 1.8 8.9 ± 1.7Mg 3.5 ± 0.5 3.5 ± 0.1Mn 1.8 ± 0.6 8.5 ± 2.1N 20.0 ± 2.6 24.0 ± 0.6P 2.6 ± 0.5 1.9 ± 0.7S 3.7 ± 0.4 2.9 ± 0.3Ca/N 102.6 ± 20.8 52.5 ± 15.5K/N 17.7 ± 4.2 14.6 ± 0.8Mg/N 68.2 ± 6.9 37.1 ± 6.4P/N 13.2 ± 2.2 8.1 ± 2.9

1Crumbled leaf subsamples were digested in concen-trated HNO3. The concentrations of the elementswere then determined using an inductively coupledplasma analyzer (Perkin Elmer, Norwalk, USA).

Table 1. Concentration of chemical elements1 in theartificial peat–sand mixture and in the acid forest soilafter sterilization at 122°C (1.1 bar) for one hour.

Element Peat–sand Forestmixture soil

Ca (µg g–1) 1228 31K (µg g–1) 109 20Mg (µg g–1) 113 6Mn (µg g–1) 9.3 113N (mg g–1) 0.9 1.4P (µg g–1) 33 39

1Concentrations of base cations and Mn were deter-mined by an inductively coupled plasma analyzer(Perkin Elmer, Norwalk, USA) after extraction of 20g of soil in 100 ml 0.1 M BaCl2 for two hours, whileP was extracted according to the Bray method. TotalN was analyzed using the Kjeldahl technique (Anon-ymous, 1998). The analyses were based on five sam-ples for each soil type.

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metabolites, since a high uptake of these nutri-ents will result in an increased metabolization forgrowth, thus depleting the carbohydrate pools.Roots may therefore have less protection againstpathogen infection and subsequent damage.

In the peat–sand mixture, on the otherhand, N is likely to be limiting, since the soiland leaf concentrations are relatively low com-pared with other nutrients (Tables 1 & 2). If agrowth nutrient is limiting, the available miner-al is likely to be metabolized in the roots firstdue to the close proximity between the sites ofuptake and mineral assimilation (Ericsson et al.,1996). Consequently, less carbohydrates will bemetabolized in the shoot and shoot growth willbe reduced. A larger fraction of carbohydrateswill be transported to the roots and thus beavailable for metabolization in the root, andsubsequently root growth. Furthermore, therelatively high availability of non-structural car-bohydrates is likely to lead to an increased flowthrough pathways that result in the synthesis ofcarbon-rich secondary metabolites (Lambers etal., 1998), some of which may be used to re-strict root infection by Phytophthora. The seed-lings grown in the peat–sand mixture maytherefore have a better protection of their rootsand they may also be able to maintain a high

root production, replacing the roots that are lostdue to infection by P. quercina. The greater totalbiomass (Figure 6a) and the significantly higherroot:shoot ratios (Figure 6b) of seedlings grownin the peat–sand mixture (control as well as in-fected) compared with seedlings grown in theforest soil support this hypothesis. Measuringthe concentrations of some defence compoundsmay confirm the hypothesis.

Differences in carbon allocation patterns intrees, as a consequence of soil chemistry, is also alikely reason for the lack of difference in livefine-root length between mature oaks in standsinfested with P. quercina and those growing in acorresponding stand where the pathogen wasnot present (Paper IV). The concentrations of Pin the organic layer and in the mineral soil werelower in several of the stands where P. quercinawas present than in the corresponding standwhere the pathogen was not present. In accord-ance with the suggested mechanism in Figure 5,low availability of P is likely to lead to a highallocation of carbohydrates to root growth.Subsequently, the trees may replace the rootsthat are lost due to Phytophthora infection andmay also have a high allocation of carbohydratesto defence. This is supported by the significantcorrelation between the concentration of P in


Figure 6. a) Total biomass and b) root:shoot ratios of control seedlings and Phytophthora-infected seedlingsgrown in an artificial nutrient-rich peat–sand mixture and in an acidic, N-rich forest soil with low concentra-tions of base cations. Statistics given are for soil type for two-way ANOVA. There were no significant differenc-es between control and infected seedlings. C = control seedlings; 1 = seedlings infected with P. quercina isolate1; 2 = seedlings infected with P. quercina isolate 2; P = peat–sand mixture; F = forest soil.

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the leaves and the live fine-root length (Pearsoncorrelation: rho = –0.77, p = 0.025, n = 8). Inthe severely declining trees, the continuous pro-duction of new roots have probably depletedthe carbohydrate stores, and the production ofnew carbohydrates is obviously too low to com-pensate for the loss of roots, resulting in an im-balance between root death and root replace-ment, thereby enhancing tree decline (see alsoChapter 7; Figure 7).

These results suggest that when oak growthis limited by N or P, more carbohydrates will beavailable for root regeneration and the produc-tion of defence compounds unless the deficien-cy is severe. If trees are limited by K, Mg or Mn,the amount of carbohydrates available for de-fence and root production will be relatively low,and rootlet death caused by Phytophthora mayexceed rootlet replacement by the trees, thusweakening the plants and enhancing the ability

of the pathogen to invade the roots. Acidifica-tion-induced reductions in soil-available K andMg, and a high N deposition (see also Chapter7), may therefore accelerate the damage causedby P. quercina on root systems of oak in south-ern Sweden. The influence of soil type and soilcondition on symptom expression and diseaseincidence is well-known in little leaf disease ofshortleaf pine (Pinus echinata), caused by P. cin-namomi in the south-eastern USA (Oak &Tainter, 1988; Tainter & Baker, 1996) and inNew Zealand (Newhook & Podger, 1972).

7. A Conceptual Model for theDisease Development in OakBased on the literature presented in Chapter 5,describing how different abiotic and biotic fac-tors affect Phytophthora species and the inter-


Figure 7. A conceptual model of how different abiotic and biotic factors influence the aggressiveness of P.quercina, the susceptibility of Q. robur and the subsequent disease development. The variable “Phytophthoraactivity” includes processes that affect the capacity of the pathogen to infect roots (such as zoospore production,dispersion, taxis, adhesion and cyst germination); CH = carbohydrates. Note that arrows only indicate thedirection of the causality. An arrow with a positive causality sign implies that a change in the affecting factordrives a change in the affected factor in the same direction. Arrows with negative causality signs imply a changein the opposite direction. Thick arrows indicate major feedback loops in the system. For further informationand a description of the causal loop diagram, see Chapter 7.

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action between the pathogens and their hosts, aconceptual model was constructed (Figure 7).The aim of this simplified model was to give anoverview of how the different abiotic and bioticfactors act simultaneously on the pathogen–host system, sometimes accelerating disease de-velopment, and sometimes hindering it. Themodel is applicable for the interaction betweenPhytophthora species and Quercus species grow-ing in acid forest soils in a temperate climate(similar to southern Swedish conditions). Onlyeffects of Phytophthora on the fine-root systemhave been considered. For a more thorough de-scription of the C allocation in trees, see Chap-ter 6 (Figure 5). Note that the variable “micro-bial activity” does not include pathogens, sincethese are considered separately, and that the ar-rows in the causal loop diagrams show only thedirection of the causality. An arrow with a posi-tive causality sign implies that a change in theaffecting factor drives a change in the affectedfactor in the same direction (i.e. an increaseleads to and increase, and a decrease leads to adecrease). Arrows with negative causality signsimply a change in the opposite direction (i.e. anincrease leads to a decrease, and a decrease leadsto an increase). Temperature has not been in-cluded in the figure, since it is likely to influencePhytophthora activity, microbial activity andphysiological processes in the tree in a similardirection. Note that increased soil moisturedoes not necessarily imply increased uptake ofwater. However, the link has been includedsince a low availability of water will lead to areduced water uptake.

The causal loop diagrams in Figure 7 can beexplained as follows. High soil moisture, as aresult of heavy rain or prolonged periods withrain, will increase the production and disper-sion of Phytophthora zoospores (the infectingunit), thereby increasing the probability of rootinfection and consequently also root damage.Root damage may lead to an increased exuda-tion of carbohydrates from the roots, therebyfacilitating zoospore taxis of Phytophthora (Car-lile, 1983). When the zoospores can find theroots easily, this is likely to result in a higher de-gree of infection and thus an increase in theamount of damage. A high amount of rootdamage results in a reduction in the amount of

live fine-root length, unless high production re-places the roots that are lost. If the live fine-rootlength is decreased, the uptake of water and nu-trients will be impaired. Shortage of nutrientsthat are important in photosynthesis will leadto reduced C fixation, and subsequently re-duced tree vitality (see Chapter 6; Figure 5).However, increased root exudation may alsoenhance the microbial activity. This increase inmicrobial activity may reduce the root infec-tion by Phytophthora, since Phytophthora spe-cies usually are weak competitors (Tsao, 1990).Flooding of soils for a long period of time mayresult in anaerobic conditions in the soil, whichmay affect the activity of the soil microbialcommunity (including mycorrhizal fungi) neg-atively, thereby increasing the probability ofPhytophthora infection of roots. Furthermore,the lack of O2 may cause direct damage to theroots, although Q. robur is believed to be rela-tively tolerant to flooding (Colin-Belgrand etal., 1991).

Low soil moisture, or drought, has the re-verse effect on sporangial production and thedispersion of zoospores, and may thus decreasethe degree of root infection. However, droughtalso influences the soil microbial communitynegatively, thereby increasing the potential forPhytophthora to infect roots in the event of asubsequent increase in soil moisture, since thesepathogens respond extremely quickly to such anincrease. Furthermore, severe drought may de-crease the rate of photosynthesis (Horner, 1990;see also Section 5.1.1), and subsequently im-pede the replenishment of the carbohydratepools in the plant. If the pools of carbohydratesdecrease, the allocation of C to the productionof defence compounds decreases, and this mayincrease the ability of Phytophthora to infectroots in the event of an increase in soil moisture,and render tissue more susceptible to damageonce infection has taken place.

Accumulation of N in the soil may influencethe development of the disease through the pos-itive effect of nitrate on zoospore production. Inaddition, high uptake of N is likely to increasethe allocation of C to growth, particularly shootgrowth (Marschner, 2003), and subsequentlydecrease the relative amount of C available forroot growth and the production of secondary


32

compounds (see also Chapter 6; Figure 5). Roottissue may thereby become more susceptible toroot infection and damage. Low C allocation toroot growth may also lead to a reduced ectomy-corrhization and growth of external mycelia(Brunner, 2001), thereby increasing the poten-tial for Phytophthora to infect roots. Other soilchemical characteristics may also influence dis-ease development (see Section 5.1.2). Low con-centrations of Ca, high concentrations of Aland low pH all influence zoospore productionnegatively, thereby decreasing the probability ofroot infection. Low concentrations of Ca mayalso influence zoospore adhesion to solid surfac-es, zoospore taxis and oospore germination neg-atively. However, low concentrations of Ca mayalso increase the susceptibility of the plant toroot infection, due to the necessity of Ca forstructural growth in cell walls and plasma mem-branes (Marschner, 2003). In addition to influ-encing zoospore production negatively, low pHand high concentrations of Al are known tohave negative effects on root elongation and celldivision (Marschner, 2003; Puhe, 2003) and toinfluence the microbial activity negatively. Re-garding the influence of other nutrients (K,Mg, P), which have no effect, or variable ef-fects, on Phytophthora, but may influence the Callocation patterns in plants, see Chapter 6(Figure 5).

Secondary pathogens often follow infectionof roots by Phytophthoras, and may exacerbatethe root damage, thus increasing the carbohy-drate exudation from the roots, and, may there-by influence the activity of Phytophthora as wellas other microbes. Decreased tree vitality (de-fined as increased crown defoliation) will resultin a reduction in photosynthesis, thereby reduc-ing the pool of available carbohydrates. Subse-quently, less carbohydrates will be available forgrowth and defence.

In conclusion, P. quercina has the ability toreduce the live fine-root length of oak substan-tially, but as long as the tree can sustain newroot production to replace the lost roots, nutri-ent and water uptake will not be affected.However, continuous root infection may grad-ually deplete the carbohydrate stores, and lesscarbohydrates will then be available for rootand shoot growth and defence. The susceptibil-

ity of the tree to pathogen infection as well as toother types of stress will thus increase. If envi-ronmental conditions are favourable for thepathogen, the live fine-root length is likely toeventually decrease. A reduced fine-rootlength, together with the infection of coarserroots, will result in a lower capacity for uptakeand transport of water and nutrients. This mayresult in a reduction in the rate of photosynthe-sis, and smaller amounts of carbohydrates willthus be available to regenerate the root system,as well as for the production of secondary me-tabolites. Subsequently, tree vitality will de-crease, as disease development is acceleratedthrough negative feedback loops. The range offield responses to Phytophthora, from slow de-cline to sudden death of trees, is likely to reflectdifferences in the interaction between the path-ogen and the host, as a consequence of theirresponse to the different abiotic and biotic fac-tors presented in Figure 7.

8. Oak Decline in SouthernSweden – A HypothesisThe ability of P. quercina to infect and causesubstantial root damage to both seedlings andmature trees in forest soils under varying envi-ronmental conditions suggests that this patho-gen contributes to oak decline in southern Swe-den. Since both pathogen aggressiveness andhost susceptibility are strongly influenced byvarious abiotic and biotic factors, a combina-tion of events is probably necessary for declineto occur as a consequence of Phytophthora infec-tion. Severe flooding, drought, low availabilityof K and Mg in the soil or high availability of Nmay all enhance the negative effects of P. querci-na (see Chapter 5 & 7). However, many of thesites from which Phytophthora species were notrecovered also show severe symptoms of crowndamage. This suggests that the decline of oaksin southern Sweden cannot be attributed to thissingle factor.

During the past decades, an extensive acidi-fication of southern Swedish forest soils hastaken place, leading to a diminishing nutrientcapital, particularly of base cations, in the soil(Falkengren-Grerup, 1987; Falkengren-Grerup


33

et al., 1987; Tamm & Hallbäcken, 1988). A lowavailability of base cations, or other nutrients,might at elevated concentrations of N, result innutrient imbalances in the tree (Nihlgård,1985; Thelin, 2000). As a consequence, tree vi-tality may decrease. In the oak stands investi-gated in this work, no significant differences insoil acidity, the concentration of base cations orthe concentration of N in the soil were foundbetween healthy and declining trees, or be-tween healthy and declining stands (Papers IV& V). Instead, leaf chemical analysis and thepatterns of root growth indicated that treeswere N or P limited (Paper IV). However, alarge-scale survey of 109 oak stands in southernSweden showed that stands growing in soilswith very low base saturation values (<10%)had significantly higher crown defoliation thanstands growing in soils with normal base satu-ration values (>20%; Sonesson & Anderson,2001). Oak vitality therefore seems to be relat-ed in some way to nutrient availability in thesoil, at least in certain regions of southern Swe-den. Possibly, the low availability of base cati-ons have led to a decrease in the C fixation ofthe trees (see Chapter 6; Figure 5), which mayhave caused a reduction in the production ofsecondary metabolites in the trees, thereby pre-disposing them to factors such as pathogens orfrost. Considering the low concentrations ofbase cations in the oak forest soils in southernSweden, continued deterioration of this cationpool seems likely to further destabilize the oakecosystems.

Another factor that is probably involved inoak decline is climate (Barklund & Wahlström,1998; Paper V). Severe bark necrosis, due tofrost damage caused by the unusually cold win-ters at the end of the 1980s was suggested to bethe initial cause of oak decline in southern Swe-den (Barklund & Wahlström, 1998; Barklund,2002). In Paper V, significant positive correla-tions are reported between the average crowndefoliation as well as the proportion of damagedtrees and average summer precipitation and av-erage annual precipitation for the years 1984 to1999 in 21 oak stands in the southernmost partof the country. The interpretation of the data iscomplicated, since many different factors maybe correlated with precipitation, and the meas-

urements of precipitation were only rough esti-mates, but, together with the study by Barklund& Wahlström (1998), the results indicate thatextreme weather events during the 1980s and1990s, with fluctuations between years with ex-ceptionally high precipitation, years with hot,dry summers and years with low winter temper-atures, may have affected the trees negativelyand predisposed them to the action of other abi-otic and biotic stress factors.

Several studies carried out in central Europehave emphasized the necessity of defoliators foroak decline to occur (e.g. Thomas et al., 2002).The most important defoliators of oak in Swe-den are Tortrix viridana, Erannis defoliaria andOperophthora brumata (Barklund, 2002). Re-peated defoliation by these insects is likely toresult in the depletion of carbohydrate stores,followed by reduced growth and reduced pro-duction of secondary metabolites. Thus, thesusceptibility of trees to other stress factors willincrease. Attack by the ascomycete Microsphaeraalphitoides on the second flush of leaves mayfurther increase the susceptibility of the tree. Inlater stages of decline, when the tree is alreadyseverely weakened, several different secondarypathogens may infect the tree, occasionallycausing the death of the tree. Apart from thefactors already mentioned, forest managementis likely to be involved in the decline process ofoaks through attempts to achieve high produc-tion at sites that are not especially suitable foroak, or by neglecting to perform measures nec-essary to support good growth.

Based on the research hitherto performed insouthern Sweden, it appears that several differ-ent factors may be involved in oak decline. Insome cases, one single factor may be responsiblefor the decline at a particular site. However, thenonspecific symptoms of disease in trees, andthe inconclusive results when attempting tofind causal factors, suggest that at most sites,several of these factors interact with each other,causing a complex development of disease. Cli-matic fluctuations, low availability of base cati-ons and forest management probably act as pre-disposing factors, increasing the susceptibilityof the tree to the actions of inciting factors suchas Phytophthora species and defoliating insects.After the tree has become substantially weak-


34

ened, secondary pathogens may infect the trees,thereby contributing to the decline, and occa-sionally causing death of the tree.

A conceptual model describing how the fac-tors discussed above (infection by P. quercina,low availability of base cations in the soil, cli-matic extremes, defoliatiors and secondarypathogens) may affect oak vitality and how theymay interact with each other in causing declineof oaks in southern Sweden is shown in Figure8. I would like to emphasize that the model de-scribes a possible chain of events and is only in-tended as a starting point for further discussion.More thorough investigations are needed to de-termine the importance of the factors included,and to evaluate whether there are other causalfactors that are not yet described. For visibility,only the most prominent links between factors

are included. For more information regardingPhytophthora activity, tree susceptibility and Callocation in trees, see Chapters 5, 6 and 7.Note that increased soil moisture or availabilityof base cations will not necessarily result in anincreased uptake of water and cations. Howev-er, the links have been included since low avail-ability of water or base cations will lead to re-duced uptake. The model can be explained asfollows. If defoliating insects attack a tree, theamount of foliage will decrease, with a subse-quent decrease in the ability of the tree to pho-tosynthesize. New foliage will therefore have tobe produced using stored carbohydrates. If de-foliation is repeated, the carbohydrate stores arelikely to become depleted. Together with the re-duction in leaf area, this will reduce the amountof carbohydrates available for the production of


Figure 8. A conceptual model of how the different factors hypothesized to be involved in southern Swedish oakdecline may interact with each other and affect tree vitality. The suggested causal factors are indicated with aframe. The variable “Phytophthora activity” includes processes that affect the capacity of the pathogen to infectroots (such as zoospore production, dispersion, taxis, adhesion and cyst germination); CH = carbohydrates;photosynthesis nutrients = K, Mg, Mn. Note that arrows only indicate the direction of the causality. An arrowwith a positive causality sign implies that a change in the affecting factor drives a change in the affected factorin the same direction. Arrows with negative causality signs imply a change in the opposite direction. For furtherinformation and a description of the causal loop diagram, see Chapter 8.

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secondary metabolites, thus increasing the sus-ceptibility of the tree to other stress factors, suchas pathogens or frost. Severe drought may alsoreduce the rate of C fixation through its nega-tive influence on the stomatal conductance ofleaves (Kramer, 1983; Horner, 1990). Less car-bohydrates will then be available for growth anddefence. Water fluctuations in the soil, such asalternating periods of flooding and drought,may enhance the effects of a prospectivedrought on the tree by causing the oaks to de-velop a more shallow root system (Oosterbaan& Nabuurs, 1991). If Phytophthoras are presentin the soil, the production of zoospores will in-crease rapidly when soil moisture increases aftera drought. The ability of Phytophthora to infectroot tissue will then be high, at the same time asthe ability of the tree to defend itself and replaceroots lost due to pathogen attack will be low. Areduction in the amount of live fine roots willimpede the ability of the tree to take up waterand nutrients, and may thereby affect the C fix-ation negatively. Apart from defoliators and soilmoisture, shortage of certain base cations mayalso affect photosynthesis negatively, since Kand Mg are important for stomata regulation,capture of light energy and functioning of en-zymes central to carbon fixation (Ericsson et al.,1996; Marschner, 2003; Figure 5). Frost maycause necrosis of both trunks and roots, therebypossibly reducing the water and nutrient up-take, and also providing a gateway for second-ary pathogens.

In conclusion, all the factors suggested to beof importance in southern Swedish oak declineare likely to eventually influence the produc-tion of carbohydrates, and thereby the abilityof the tree to replace lost tissue and to producesecondary metabolites. The susceptibility ofthe tree to the other stress factors will thus in-crease, and tree vitality will decrease. The rateof tree decline will vary depending on howmany of these factors are acting on the tree si-multaneously, and on how they interact withother abiotic and biotic factors present at a site.With regard to P. quercina, low availability ofcertain base cations in the soil, periods ofdrought or excessive flooding, and defoliationby insects, are likely to accelerate the develop-ment of disease.

9. ConclusionsBased on the results presented in this thesis, Ihave drawn the following conclusions.(i) Phytophthora species are present in south-

ern Swedish oak stands. The most fre-quently recovered species is the oak-specificP. quercina. The oak forest soils in whichPhytophthora species occurred had a some-what higher rhizosphere pH and a higherbase saturation than the forest soils inwhich the pathogens were not present.

(ii) The southern Swedish isolates of P. queci-na can infect roots and cause damage tofine roots as well as coarser roots of Q.robur seedlings in acidic forest soils undera mesic water regime. Substantial damagewas caused both in the presence and in theabsence of the inherent soil microflora. Incontrast to what was expected, the patho-gen caused more damage to the root sys-tem of seedlings grown in an acid forestsoil than to seedlings grown in a nutrient-rich artificial mixture of peat and sandwith a higher pH. It is hypothesized thatthe more severe damage in the forest soil isdue to low availability of certain base cati-ons (K, Mg) in relation to N in this type ofsoil. A low availability of these cations mayreduce the rate of photosynthesis of seed-lings and may also affect the allocationpatterns in the plant, thereby reducing theseedlings’ ability to produce defence com-pounds and to replace roots that are lostdue to pathogen infection.

(iii) P. quercina can infect and cause significantdamage to root systems of mature trees.The effect of the pathogen seemed to de-pend on tree vitality and climatic condi-tions. In stands without P. quercina, thelength of the fine-root system did not differbetween trees of differing vitality, demon-strating that fine-root decay does not nec-essarily occur prior to noticeable above-ground symptoms in oaks.

(iv) Although a significant reduction in livefine-root length of seedlings infected withP. quercina was observed, no significant ef-fects on above-ground growth or on leafnutrient concentration could be detected


36

after the short-term greenhouse experi-ments. There were also few differences inleaf nutrient concentration between ma-ture oaks of differing vitality. This is con-sistent with previous studies, suggestingthat root rot must usually be severe beforeabove-ground effects in trees can be de-tected.

(v) A weak association was found between vi-tality of oak stands and occurrence of P.quercina, indicating that oak stands weremore likely to be declining if P. quercinawas present in the soil.

The observation that P. quercina causes sub-stantial root damage to both seedlings and ma-ture trees in forest soils suggests that this patho-gen is involved in oak decline in southern Swe-den. The continuous replacement of roots lostdue to pathogen infection is likely to graduallydeplete the carbohydrate reserves of the tree,and may subsequently result in a reduction inroot and shoot growth, as well as in an increasedsusceptibility of the tree to further infectionsand other types of stress. Since the effects of P.quercina on root systems of oak are highly de-pendent on the prevailing environmental con-ditions, the development of the disease is likelyto vary substantially depending on geographicallocation, season and soil type. However, undersevere environmental conditions, epidemic de-velopment with die-back of oaks seems possi-ble. In some southern Swedish oak stands, P.quercina may be the factor of greatest impor-tance in the decline of the stand. In most stands,tree decline is more likely to be due to a combi-nation of abiotic and biotic factors, which grad-ually decreases the vitality of the tree, occasion-ally resulting in its death.

10. Future PerspectivesDespite a substantial improvement in ourknowledge concerning the involvement of Phy-tophthora species in oak decline and in our gen-eral understanding of the interactions betweenthese pathogens and their hosts during the pastdecades, there are still significant gaps thatneed to be filled regarding the response of path-ogen aggressiveness, host susceptibility and

subsequent disease severity to various abioticand biotic factors. A possible reduction ofpathogen infection as a consequence of exten-sive mycorrhizal colonization of roots or highmicrobial activity in the soil remains to be con-firmed for Phytophthora species and oaks. Thisis highly interesting when considering oak de-cline, since some studies have indicated re-duced mycorrhizal colonization of decliningoaks (Causin et al., 1996; Kovacs et al., 2000),thereby possibly enhancing their susceptibilityto Phytophthora infection. Since the response todifferent abiotic factors may vary depending onthe specific species combination, supplementa-ry studies on how increasing concentrations ofsoil N and Al and decreasing soil concentra-tions of base cations affect the aggressiveness ofP. quercina and, equally important, the suscep-tibility of Q. robur, are needed. In addition, itwould be interesting to evaluate the effects ofsoil amelioration, such as liming, on diseasedevelopment, since this kind of treatment maybe necessary in the future to improve soil qual-ity in the areas most exposed to soil acidifica-tion.

Regarding oak decline in general, more stud-ies are required to evaluate the interactions be-tween several different abiotic and biotic fac-tors. Today, few studies exist in which three ormore factors included in a hypothetical modelhave been tested together. Greenhouse experi-ments on defoliation followed by drought andinfestation by Phytophthora, or a high input ofN, followed by defoliation and pathogen infes-tation, would be very interesting in order toevaluate whether symptoms similar to those ob-served in the field can be artificially induced.

Acknowledgements – This work was generouslyfunded by The Swedish Research Council for theEnvironment, Agricultural Sciences and SpatialPlanning and by CF Lundström’s Fund, The Envi-ronmental Fund of Region Skåne, Erik and EllenSökjer Petersen’s fund, AB Gustaf Kähr, Gustafs-borg Säteri, the National Board of Forestry, the Re-gional Forestry Board Södra Götaland, the RegionalForestry Board Östra Götaland, the ConsolidatedFunds of the Royal Swedish Academy of Agricultureand Forestry, Skogssällskapet, SparbanksstiftelsenKronan and Tarkett Sommer.


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To my family…

Tack mamma och pappa för allt stöd ni givit mig, för att ni alltid ställt upp för mig vad jag än velatgöra, för att ni trott på mig och för att ni lärde mig att ingenting är omöjligt. Ni är de bästa föräldrarman kan önska sig! Tack Mia, min kära syster, för att du också alltid funnits där för mig och för attdu tagit så god hand om mig. Vi har gjort det mesta tillsammans och jag hoppas att vi ska fortsättagöra saker tillsammans och ta hand om varandra! Anton – ingen kan få mig att skratta såsom du kan!Jag älskar er!

To all the people who made this thesis what it is…

Ulrika – you are the supervisor all PhD-students dream of! My time at the Department has been apleasure thanks to you. You have given me excellent feedback on all the papers included in this thesisand without you there would be no papers, nor a thesis. You always see things from the bright sideand you make the most complicated things seem very simple; and somehow everything alwaysworks out. Thank you for always believing in me and thanks for being my friend.

Bengt – thank you for always making time for me when I needed it, and for always having ideas,suggestions or a gut feeling that helps. The South African excursion that you organized was thehighlight of my PhD-education. I had a lot of fun on that trip!

Thomas – thank you for teaching me the technical skills required to perform the research that I havedone, and for teaching me how to identify Phytophthora species. My week in Freising at the begin-ning of my PhD-studies was very valuable. You have always read the manuscripts very carefully andgiven me many useful comments.

Ursula – thank you for supporting me and helping me in times of crisis.

Anders J – I will always be grateful for all the digging you have done, and for your never-endinggood humour. Even after four days spending 14–15 hours per day digging and sieving roots you arestill happy! Thanks for all the interesting talks about training and competition that made the fieldwork much easier and nicer.

Jimmy – thanks for helping me with the field work and for being my friend. You always entertainedme with nice stories from our youth during field work and you gave me the best laugh ever whenyou ran like a maniac trying to escape a herd of very, or only slightly, aggressive cows (depending onwho is telling the story!) in our sampling plot.

To all the lab assistants – Ragnhild, Maj-Britt, Maj-Lis, Anita and Irene, as well as Tommy, thanksfor analysing all the samples so quickly and efficiently. Special thanks to Ragnhild and Maj-Britt forwashing an awful lot of roots for me. I know it is boring, but you did an excellent job! Ragnhild –thanks for introducing me to the lab, for always making sure I found everything I needed and foralways fixing everything. And thanks for calling just for a chat late in the evening when I was alonein the office. Thank you Bengt J and Rune for your technical support in the greenhouse.

Thank you!

44

Håkan W – thanks for taking the time to read the manuscripts, and for your valuable commentswhich improved them substantially.

Stefan Anderson (SVSSG) and Martin Andersson (SVOG) provided me with data for Paper V andhave answered all kinds of questions. Thank you for your time. Thank you Stefan for teaching mehow to estimate crown defoliation and how to recognize different types of oak damage. GustafFredriksson has generously helped in raising financial support for the initial part of the PhD project.Helen Sheppard has corrected the English in most of the papers and in the thesis – thanks for yourskilful help and for the many suggestions that made the papers so much better. Special thanks toGunilla Anderson for putting all the pieces together into a thesis.

To the people at work who made me feel good…

Hans – We have done quite a lot together now. We have shared the office for two years (or maybeeven more) and we have been to South Africa and Australia together. They say you never really knowsomeone for real unless you travel together… We survived it! You always have something to tell andyou can for some reason mend all the machines that I break. Thanks for all your help; I am going tomiss sharing office with you!

Ann-Mari, Carna, Gunnar, Katarina, Jacqueline, Pernilla, Lena, Maria – it is/was nice to have youaround. You create a positive atmosphere in the office. Thanks to all of you for always having timefor a chat and a laugh.

To my friends…

Anna och Terese – ni är mina äldsta och bästa vänner! Tack för att ni alltid finns där även om min tidinte alltid räcker till för att vara den vän som jag vill vara. Förhoppningsvis ska det bli bättre nu…

Beatrice – you are an outstanding person! I have never met anyone like you and I do not think I everwill. Thanks for staying with me and Salim and making everyday life into an adventure. I miss yourcheerful spirit.

Marta och Chéché – ni är de varmaste och hjärtligaste människor jag någonsin träffat. Och ni hardet mysigaste cafeét i stan – en tillflyktsort undan alla bekymmer. Jag mår alltid så mycket bättreefter en fika hos er!

Sist men inte minst…

Salim – you are my best friend, my soulmate! Thank you for your unconditional love, for alwaysbeing there for me, for supporting me, for putting up with the long working hours, for helping mewith the papers, for coping with my strange quirks, for always sorting out all the difficulties and formaking me a better person. I love you more than I can ever explain. You have made me realize whatlife is all about!

phytophthora and oak decline - impact on seedlings and ... · 4.1 an introduction to the genus...

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