1

Chapter 1.

Literature Review and Research Objectives

ABSTRACT

With increasing environmental regulations focused on water quality, recycling irrigation water is

being adopted by the nursery and greenhouse industries as a relatively simple method to avoid

release of nutrient and pesticide wastes. This method also conserves often expensive and/or

scarce water resources. The occurrence of Phytophthora and Pythium spp. in irrigation water has

been documented in diverse geographic and agricultural regions. These two genera include many

plant pathogens. Traditional techniques for assaying water for these organisms include baiting,

filtering, and centrifugation, which employ selective media for detection. Novel methods for

detection have not been investigated thouroughly. The Pythiaceae are well adapted to an aquatic

environment and may be spread through irrigation water. This possibility is a serious concern to

horticultural operations. Disinfestation of recycled irrigation is most commonly accomplished by

chlorination, but other methods have been investigated. In this work, locations and seasonal

fluctuations of Phytophthora and Pythium spp. in a recycled water irrigation system at a

container nursery in Virginia were determined. A culture collection of these organisms was also

established for future investigations. Additionally, Phytophthora spp. were identified and

selected isolates tested for pathogenicity in greenhouse. The effects of irrigation with naturally

infested water were assessed.

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IMPORTANCE AND POTENTIAL RISKS OF RECYCLING IRRIGATION WATER

Economic and environmental impacts of recycling irrigation water. Since enactment

of the Water Pollution Control Acts in 1972, more stringent standards of water quality have

forced many horticultural enterprises to limit pollution (Skimina, 1992). Skimina proposed that

recycling irrigation water was the most efficacious remedy to avert irrigation runoff and potential

regulatory problems. Recycling nursery effluent is an effective and easily implemented solution

for controlling runoff in nurseries compared to other complex management solutions (Wilson et

al., 1998). The main benefit of recycling water is the reduction in nutrient and pesticide

pollution, but the accompanying potential for spread of plant pathogens is a deterrent for many

operations (Lauderdale and Jones, 1997; Wilson et al., 1998).

Recycling nursery effluent involves catching runoff in ditches, French drains, or other

retention devices. Effluent water drains down a slope and eventually collects in a holding pond

located at the lowest elevation in the nursery or is pumped there. Particulars vary with individual

site topography. This captured water is mixed with fresh water before application to crops to

improve its quality (Wilson et al., 1998).

Potential risks of plant pathogens in recycled water. Thomson and Allen (1974)

outlined the threat from Phytophthora spp. in irrigation water as follows: 1) recycled irrigation

water is a source of contamination for susceptible plants and may place contaminated water in

proximity to roots and foliage; 2) Phytophthora spp. are well-adapted to an aquatic environment;

3) species of Phytophthora are primarily pathogens of plants; and 4) inoculum in irrigation water

can infest clean crops or cropping areas.

Zoosporic "fungi" are the most common "fungi" occurring in water (Baker and Matkin,

1978). A variety of species of Phytophthora (Ali-Shtayeh and MacDonald, 1991; Bewley and

3

Buddin, 1921; Lauderdale and Jones; 1997; Klotz et al., 1959; MacDonald et al., 1994;

McIntosh, 1966; Oudemans, 1999; Pittis and Colhoun, 1984; Taylor, 1977; Thomson and Allen,

1974; Whiteside and Oswalt, 1973; Wilson et al., 1998; von Broembsen, 1984), Pythium (Gill,

1970; Pittis and Colhoun, 1984; Shokes and McCarter, 1979), and other plant pathogens have

been recovered from irrigation water (Bewley and Buddin, 1921; Grech and Rijkenberg, 1992;

Heald and Johnson, 1969; Shokes and McCarter, 1979; Steadman et al., 1975; Thomson and

Allen, 1974). Phytophthora spp. recovered include: P. cactorum (McIntosh, 1966), P. cambivora

(McIntosh, 1966), P. cinnamomi (MacDonald et al., 1994; Lauderdale and Jones, 1997;

Oudemans, 1999; Wilson et al., 1998), P. citricola (MacDonald et al., 1994; McIntosh, 1966;

Wilson et al., 1998); P. citrophthora (Ali-Shtayeh and MacDonald, 1991; Klotz et al., 1959,

MacDonald et al., 1994; Thomson and Allen, 1974; Whiteside and Oswalt, 1973; Wilson et al.,

1998) P. cryptogea (Ali-Shtayeh and MacDonald, 1991; Bewley and Buddin, 1921; Lauderdale

and Jones, 1997; MacDonald et al., 1994; Taylor, 1977; Wilson et al., 1998), P. gonapodyides

(Pittis and Colhoun, 1984), P. megasperma (MacDonald et al., 1994; McIntosh, 1966;

Oudemans, 1999), P. nicotianae var. parasitica (Ali-Shtayeh and MacDonald, 1991), P.

palmivora (Ali-Shtayeh and MacDonald, 1991), P. parasitica (Bewley and Buddin, 1921; Klotz

et al., 1959; Lauderdale and Jones, 1997; MacDonald et al., 1994; Thomson and Allen, 1974;

Wilson et al., 1998), and P. syringae (Klotz et al., 1959). P. cinnamomi has also been recovered

from streams and rivers in Hawaii, Eastern Transval, and in the South-Western Cape Province of

South Africa (von Broembsen, 1984). P. cryptogea was isolated frequently from surface waters

used to irrigate citrus in the West Bank of Jordan (Ali-Shtayeh and MacDonald, 1991; Taylor,

1977).

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Phytophthora-infested irrigation water has been implicated in outbreaks of plant disease.

In the early part of the 20th century the mycologists Bewley and Buddin (1921) were among the

first workers to recognize a relationship between water contaminants and plant disease. Their

interest was precipitated by an epidemic of damping-off of tomato seedlings, which was caused

by Phytophthora cryptogea transported through irrigation water. They used a primitive filtering

apparatus and nonselective media to characterize plant pathogens present in water. In 1970 a

serious, but localized, epidemic of brown rot affected a grove of grapefruit trees in Florida. This

outbreak began within two weeks after overhead irrigation had been applied. In 1972 another

incidence of brown rot occurred, which also followed an application of overhead irrigation

water. The fungi isolated from the diseased fruit in both outbreaks were identified consistently as

Phytophthora citrophthora. However, soil baited from the grove yielded only P. parasitica (=P.

nicotianae). The source of the irrigation water was tested by Whiteside and Oswalt (1973) for the

presence of Phytophthora species. Baits of calamondin, grapefruit, lemon and oranges were

placed in random locations along the waterway. Soil samples from the water’s banks were also

sampled and baited. P. citrophthora was isolated from both the soil sampled and the baits in the

waterway. This was the first documentation of a brown rot epidemic precipitated by infested

irrigation water. This was presumed to be an isolated incident, caused by the presence of a

virulent strain and favorable environmental factors. The strain was not able to establish itself in

the environment of the soil in the grove.

Shokes and McCarter (1979) demonstrated that water infested with plant pathogens is a

serious factor limiting the efficacy of fumigation treatment in vegetable transplant production in

Georgia. They concluded that members of the Pythiaceae family are the most significant

contaminants in recycled water systems due to their ability to adapt to an aquatic environment,

5

common occurrence, and capability of inciting seedling diseases and root and stem rots of a

range of plant species. Wilson et al. (1998) found that levels of Phytophthora spp. in holding

ponds used to capture and recycle nursery effluent were elevated after implementation of water

recycling in a nursery.

Grech and Rijkenberg (1992) found in greenhouse container experiments that the vigor,

shoot, and feeder root extension of seedling citrus trees was negatively affected after a one-year

period of irrigation with naturally Phytophthora-infested water compared to citrus irrigated with

chlorine-treated Phytophthora-infested water. Although root infection by Phytophthora spp.

occurred within 4 months after initiation of the experiment in the non-chlorinated water

treatment, foliar symptom expression was not evident until 8 months and shoot extension in the

non-chlorinated treatment was not significantly reduced until 12 months into the treatment.

Additionally, the dry root mass measured at 12 months was reduced by an average of 42% in the

non-chlorinated treatment compared to the chlorinated treatment. In this greenhouse experiment

significant feeder roots were lost before aboveground symptoms were apparent. A similar trial

was performed in the field, but no differences in tree growth were apparent over a 12-month

period.

DISEASE EPIDEMIOLOGY

The Genus Phytophthora. Members of the genus Phytophthora are primarily, if not

wholly, pathogenic on plants (Erwin and Ribeiro, 1996). These species are important pathogens

of agricultural plants, but may also occur in natural ecosystems (Brasier and Hansen, 1992).

Individual species of Phytophthora may be parasitic on quite narrow or very broad ranges of host

species (Erwin and Ribeiro, 1996). P. cinnamomi is reported to be pathogenic to over 900 plant

species (Brasier, 1992; Zentmyer, 1983), while P. primulae is reported on a single plant genus

6

(Erwin and Ribeiro, 1996). The described terrestrial species of Phytophthora number over fifty

(Brasier, 1992).

Phytophthora spp. are the causal agents of some of the world’s most devastating

epidemics, including potato late blight, caused by P. infestans, eucalyptus dieback in Australia,

caused by P. cinnamomi, and black pod disease on cacao, caused by both P. palmivora and P.

megakarya (Brasier, 1992; Gregory, 1983). As M. E. Gallegly noted at the Phytophthora

Workshop at West Virginia University (June, 2000), Phytophthora comprises one of the most

economically important genera of plant pathogenic ‘fungi’.

Taxonomic Status of Phytophthora. The taxonomic status of Phytophthora has changed

dramatically in recent years. Although controversy exists, most agree that the genus should be

moved from the Myceteae to a new kingdom. Formerly, the genus Phytophthora was placed in in

the subclass, Oomycetes of the Phycomycetes. Erwin and Ribeiro (1996) endorse the

classification scheme of Dick who places Phytophthora in the kingdom, Chromista, due to its

phylogeny. The kingdom Chromista is comprised of heterokont algae, diatoms, and other protists

possessing tinsel flagellae (Brasier and Hansen, 1992). The Saprolegniomycetidae,

Rhipidiomycetidae, and Peronosporomycetidae comprise the Oomycota in this scheme.

Phytophthora and Pythium are placed in the family Pythiaceae within the order Pythiales within

the Peronosporomycetidae. Dick acknowledges that due to Phytophthora’s morphological and

physiological similarities to true fungi, the genus will be, for practical purposes, treated as a

fungus (Erwin and Ribeiro, 1996). Therefore, I will use ‘fungi’ to refer to Phytophthora, as a

term of convention.

According to Brasier (1992), the genetics of Phytophthora are more closely related to

higher organisms than true fungi. Vegetative nuclei of both Phytophthora and Pythium exist

7

primarily in a diploid state, in contrast to the haploid state of true fungi (Erwin and Ribeiro,

1996). Zoosporic dispersal and oogamy are also characteristics not shared by true fungi (Brasier

and Hansen, 1992). Other characteristics that set these organisms apart include cell walls

synthesized with cellulose and β-glucans, rather than chitin, biflagellate zoospores, and lack of

sterol synthesis (Erwin and Ribeiro, 1996). These qualities dictate unique control and

management measures for these fungi.

Phytophthora and Pythium spp. share many traits, particularly their morphological

characteristics and qualities of oosporogenesis (Brasier and Hansen, 1992). Hymexazol is used in

media to preferentially select for Phytophthora spp. over faster growing Pythium spp., but some

Pythium spp. are insensitive, and a few Phytophthora spp. are inhibited by this antifungal

antibiotic (Erwin and Ribeiro, 1996); therefore, morphological characteristics are necessary to

separate these two genera. Zoospores of Pythium spp. differentiate in a vesicle extruded from the

zoosporangium, whereas zoospores of Phytophthora spp. differentiate within the zoosporangium.

Molecular data are also accumulating that support the separation of these two genera, and

monoclonal antibodies have differentiated Phytophthora spp. from Pythium spp. (Brasier and

Hansen, 1992).

Phytophthora Disease Epidemiology. Inoculum of Phytophthora spp. has the potential

to increase from very low to high levels in a period of days to weeks. This rapid increase in

inoculum is attributed to the formation of sporangia and zoospores under favorable

environmental conditions--the most significant of which is the existence of free water. Therefore,

Phytophthora diseases are usually classified as multicyclic. Zoosporangia are the typical asexual

propagule associated with Phytophthora (Erwin and Ribeiro, 1996). Zoosporangia may produce

a germ tube or form zoospores (Carlile, 1985), but release of zoospores is correlated with high

8

water potentials (Carlile, 1985; Gisi, 1983). Germinating zoospores can produce appressorium-

like structures, microsporangia, or chlamydospores (Thomson and Allen, 1976).

All Oomycetes possess biflagellate zoospores—a tinsel flagellum on the anterior portion

and a whiplash flagellum on the posterior portion. In Phytophthora spp. the length of zoospores

ranges from 9 to 15 µm. Low temperatures are associated with zoospore release in species that

infect aerial plant parts. This may be due to the longer period of persistence of water on leaves

and stems that occurs with lower temperatures, allowing for increased zoospore motility and

infection. Some species of Phytophthora produce sporangia on aerial plant parts and high

relative humidity is required for sporangia production. Other species produce sporangia in soil

and require high ambient water potentials (i.e. solutions low in solutes). Under high temperatures

or low water potentials, direct germination of sporangia through germ tubes is favored. Heavy

outbreaks of Phytophthora usually occur in environments that favor zoospore release rather than

direct germination. This demonstrates the efficacy of disease spread through the vehicle of

zoospores (Carlile, 1985).

Thomson and Allen (1976) demonstrated the infection capability of zoospores in baiting

studies with P. parasitica (=P. nicotianae). Citrus leaf pieces were floated in water over

naturally infested soil. After ten minutes of incubation, 35% of the leaf pieces became colonized

and, within sixty minutes, 95% of the leaf baits became colonized with P. parasitica.

The motility and taxis of zoospores are factors in the spread of Phytophthora diseases.

Six to ten hours can be reasonably regarded as a probable period of activity for zoospores. At

their fastest, zoospores could swim 6 miles in 10 hours, but since they change direction

frequently, approximately 6 cm is the realistic distance a zoospore would actually travel from a

starting point. Duration of motility decreases with increasing temperature (Carlile, 1985). The

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motility of zoospores allows better transport through moving water compared with non-motile

propagules (von Broembsen and Deacon, 1997). Zoospores possess both positive and negative

chemotaxis. The former allows targeting of susceptible host tissue and the latter decreases the

chance of zoospores lysis (Carlile, 1985). Chemotaxis and motility allow these infective

propagules the maximal chance of reaching a host (von Broembsen and Deacon, 1997).

Zoospores also possess a negative geotaxis, which causes zoospores to accumulate near the

surface of soil near host tissue (Carlile, 1985).

If zoospores do not lyse they will eventually encyst. Encystment can be triggered by a

range of environmental factors, including root exudates, changes in osmotic potential, or changes

in pH. During encystment flagella are shed, motility stops, and the encysted zoospore adheres to

any solid surface in proximity. If encystment occurs on host tissue, germination of the cyst can

occur in about 30 minutes; otherwise germ tube formation may be delayed by about 3 hours.

Cysts may also produce secondary zoospores. This is more likely to occur when a suitable host is

not available (Carlile, 1985).

TRADITIONAL TECHNIQUES FOR ASSAYING PLANT PATHOGENS IN WATER

Hallett and Dick (1981) noted four methods of assaying water for aquatic fungi: 1)

detection on naturally occurring substrates, 2) trapping; 3) baiting; and 4) plating. Qualitative

data may be obtained with any of these methods, but the ability to quantify populations varies

with each of these methods. Detection on naturally occurring substrates may have applications in

situations where a single, well-defined substrate is present. Trapping is useful for propagules

with distinct morphological features; however, spores of Oomycetes are not morphologically

distinct (Hallett and Dick, 1981). Baiting and plating have both been employed for studies of

Oomycetes.

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Baiting. Baiting has been used for qualitative analysis of water-borne pathogens by many

workers (Clausz, 1974; Dick, 1966; Gill, 1970; Klotz et al., 1959; Lauderdale and Jones, 1997;

McIntosh, 1966; Middleton, 1985; Oudemans, 1999; Pittis and Colhoun, 1984; Taylor, 1977;

Thomson, 1972; Thomson and Allen, 1974; Whiteside and Oswalt, 1973; Wilson et al., 1998).

Much work with baits has been done in citrus groves where Phytophthora diseases are a serious

problem. Klotz et al. (1959) used silver stage lemons to successfully isolate P. citrophthora, P.

parasitica, and P. syringae from citrus irrigation canals and reservoirs. Thomson and Allen

(1974) found citrus leaf baits to be more effective than lemons for isolation of Phytophthora

parasitica (=P. nicotianae), P. citrophthora, and an unidentified Phytophthora sp. from runoff

irrigation water in citrus groves in Arizona. Gill (1970) used rooted cuttings of various plant

species in bait floats in an irrigation pond and recovered Pythium myriotylum and an unidentified

Pythium isolate. Wilson et al. (1998) found rhododendron and lemon leaf baits to be equally

effective in Phytophthora recovery and deemed leaf baits to be “extremely sensitive” in

detection of Phytophthora spp., sometimes recovering the species when filtration was

unsuccessful. However, baits are problematic due to their unknown degree of selectivity for an

organism (Hallett and Dick, 1981).

Dilution Plating, Continuous Flow Centrifugation, and Filtration. Dilution plating

techniques have been used in isolation of aquatic fungi from water (Thomson, 1972), but have

the disadvantage of being very labor-intensive and involving a large number of plates. The

validity of extrapolating counts of propagules recovered from relatively small volumes of water

to very large volumes contained in ponds and lakes also presents difficulties (Hallett and Dick,

1981). Filtering methods may involve direct inversion of a membrane onto an agar plate after

filtering (von Broembsen and Wilson, 1998) or a membrane may be suspended in a weak agar

11

solution, agitated to release captured propagules, and the wash solution spread on agar plates

(MacDonald et al., 1994).

Concentration of water samples through filtration (Ali-Shtayeh and MacDonald, 1991;

Bewley and Buddin, 1921; MacDonald et al., 1994; Shokes and McCarter, 1979; Pittis and

Colhoun, 1984; Thomson, 1972; Thomson and Allen, 1974; von Broembsen, 1984; Wilson et al.,

1998) and constant flow centrifugation (Clausz, 1974; Hallett and Dick, 1981; Middleton, 1985)

have both been employed as a means of assaying fungal propagules from water.

The disadvantage of filtering and centrifugation methods is that some propagules are lost

during recovery (Hallett and Dick, 1981; Middleton, 1985). Clausz (1974) reported losses of up

to 40% of spores with constant flow centrifugation. Hallett and Dick (1981) were able to develop

a method of constant flow centrifugation where losses of Achlya spp. were 15% or less.

Middleton (1985) reported a minimal loss of less than 20% of propagules with continuous flow

centrifugation. Middleton also examined zoospore loss after filtration with 8 µm Millipore filters

and plating on Phytophthora-selective agar. Two isolates each of Phytophthora cinnamomi and

P. cryptogea were used and mean losses ranged from 2 to 21% for P. cinnamomi and 10 to 17%

for P. cryptogea. A limitation inherent in filtration is clogging of the filters, which limits the

volume one can process efficiently (Erwin and Ribeiro, 1996; Middleton, 1985).

Culture Plating. Until the discovery in 1960 that Phytophthora and Pythium, unlike

other fungi, were not inhibited by pimaricin, isolation of Phytophthora was much more difficult

to accomplish by direct plating and baits were commonly used. The discovery of resistance by

Phytophthora and Pythium to polyene antibiotics initiated research on selective media for these

organisms. Initial attempts used a medium high in pimaricin (100 mg/liter), but this

concentration was found to prevent germination of spores. The concentration of pimaricin was

12

decreased, resulting in the P10VP medium of Tsao and Ocana in 1969. The 10 mg/liter

concentration of pimaricin in this medium was shown by workers not to inhibit germination. The

‘V’ in P10VP represents vancomycin (antibiotic active against gram-positive and gram-negative

bacteria) and the last ‘P’ represents pentachloronitrobenzene, which is active against many fungi,

but not Phytophthora and Pythium (Erwin and Ribeiro, 1996).

P10ARP cornmeal medium is purported to be the most effective medium for isolation of

Phytophthora spp. from soil or plant tissue (Erwin and Ribeiro, 1996). Oudemans (1999)

modified this medium with 10 mg/liter benomyl to suppress contaminants in his work with

Phytophthora cinnamomi in cranberry irrigation water. P10ARP may also be amended with

hymexazol at 50 mg/liter to select for Phytophthora spp. over faster growing Pythium spp.;

hymexazol is also inhibitory to Mortierella spp. (Erwin and Ribeiro, 1996; Oudemans, 1999).

Components of P10ARP include pimaricin (10 mg/liter), ampicillin, rifampicin, and

pentachloronitrobenzene. The ampicillin kills gram-positive bacteria and rifampicin kills gram-

negative bacteria (Erwin and Ribeiro, 1996); these two antibiotics were shown by Pittis and

Colhoun (1984) to be effective in suppression of bacteria isolated from water samples.

In work with the Phytophthora-selective medium, P10VPH, Tay et al. (1983)

demonstrated that some viability of propagules is lost due to the negative effects of the

antibiotics used and noted that hymexazol has been shown to decrease germination of zoospore

cysts of P. capsici. Generally, experiments involving germination inhibition and media

components have focused on sporangia, zoospores, and chlamydospores along with colony

growth rates. Few studies on the effect of chemical amendments in relation to germination of

oospores have been undertaken, probably because of the generally unpredictable or very slow

13

germination of oospores; however, these propagules are important in population increases of

homothallic species (Erwin and Ribeiro, 1996)

Identification. Although numerous keys to the species of Phytophthora exist, keying

isolates to species is generally considered difficult due to the paucity of morphological

characteristics present on culture plates. Additionally, much “variability and overlapping

features” occur within individual species (Erwin and Ribeiro, 1996). M. E. Gallegly noted at the

Phytophthora Workshop at West Virginia University (June, 2000) that the morphological

features that distinguish Phytophthora from other fungi include: motile zoospores; differentiation

of zoospores within the sporangium and release of zoospores through the sporangial apex;

zoosporangia formed in succession at the apex of a simple branch or of a simple or compound

sympodium; persistent or non-persistent sporangia; oogonium normally with a single oospore;

and paragynous or amphigynous antheridia, usually single. Other unique physiological features

of this genus include the requirement for exogenous sterols for sporulation; exogenous source of

thiamine required for growth; light requirement for asexual sporulation, and dark requirement for

formation of sexual structures.

Colony types on agar vary from radiate to chrysanthemum, rosaceous to stellate, or

otherwise, but colony morphology is not adequate for identification. Colony patterns may also

vary within a species (Erwin and Ribeiro, 1996). The morphological characteristics used for

identification to species include: 1) sporangial papillation, shape, size, and length-breadth ratio;

2) persistent or non-persistent nature of sporangia; 3) pedicel length of non-persistent sporangia;

4) internal, external, or lack of proliferation of sporangia; 5) branching pattern of

sporangiophores; 6) occurrence or lack of chlamydospores and/or hyphal swellings; 7)

production of sexual bodies in single culture; 8) amphigynous or paragynous antheridia; 9)

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ornamentation or lack of ornamentation of oogonia and shape of oogonial stalk; and 10)

temperature parameters (Erwin and Ribeiro, 1996). Some of these characteristics are primary

characteristics used in identification while others are supporting characteristics (Waterhouse et

al., 1983).

Much work has been done to describe species and formulate keys, but until the

completion of Grace Waterhouse’s key in 1963, confidence in ‘good’ species and keys was

lacking. Waterhouse formulated six groupings of Phytophthora and described 43 species and

subspecies (Waterhouse, 1963). In 1978 Newhook, Waterhouse and Stamps, prompted by

discoveries of species and problems with the Waterhouse key, developed a tabular key

(Waterhouse et al., 1983). In 1990 this key was revised (Stamps et al., 1990). Erwin and Ribeiro

(1996) formulated a tabular key. Other keys have also been developed, which, while not as

comprehensive, are useful. These include Ho’s (1981) key to 38 species and subspecies of plant

pathogenic Phytophthora and M. E. Gallegly’s unpublished key (Phytophthora: Morphology and

Identification of Some Species; revised April 1, 2000), which includes 26 plant pathogenic

species of Phytophthora.

MODERN ASSAY METHODS

Enzyme-linked Immunosorbent Assay. Enzyme-linked Immunosorbent Assay (ELISA)

offers some advantages over traditional culture plating for detection of Phytophthora spp. The

primary advantages are the ability to detect low levels of an organism and the rapidity of the test.

ELISA kits for detection of some Phytophthora spp. are commercially available (Agri-

Diagnostic Associates, Cinnaminson, New Jersey). MacDonald et al. (1990) demonstrated

detection with ELISA kits of Phytophthora cryptogea on many ornamental plants to be

comparable to traditional isolation methods using selective media; however, it was

15

acknowledged that many unknowns still exist and more work needs to be done before

interpretations of tests with these kits can be reliably made. Ali-Shtayeh et al. (1991)

demonstrated the use of commercially available ELISA kits for identification of Phytophthora

and Pythium spp. in water. All of the Phytophthora spp. tested reacted positively with the

Phytophthora ELISA kits and had no reaction with the Pythium kits. However, all Pythium spp.

tested also demonstrated a positive reaction with the Phytophthora kits and a negative reaction

with the Pythium kits (Ali-Shtayeh et al., 1991; Erwin and Ribeiro, 1996). Cross-reactivity of

certain ELISA kits has been observed with some species of Pythium and Peronospora (Erwin

and Ribeiro, 1996). Additionally, taxonomists need to address uncertainties regarding the species

of Phytophthora and Pythium commonly occurring in nurseries, so that reactions with specific

antibodies can be analyzed (MacDonald et al., 1990; Erwin and Ribeiro, 1996). ELISA kits for

the detection of citrus pathogens in citrus groves have been reported to be useful and reliable

(Erwin and Ribeiro, 1996).

DNA Probes. Relatively recent attempts have also been made to use DNA probes to

increase the reliability, sensitivity, and rapidity of identification of species of both Pythium and

Phytophthora. Judelson and Messenger-Routh (1996) successfully quantified growth of

Phytophthora cinnamomi in avocado roots with a species-specific DNA probe, which did not

cross-hybridize with DNA of other Oomycetes, fungi, or plants. Goodwin et al. (1989, 1990)

used DNA probes to identify both P. parasitica (1989) and P. citrophthora (1990); these probes

did not cross-hybridize with other Phytophthora or Pythium spp. (Erwin and Ribeiro, 1996;

Goodwin et. al, 1989, 1990). Lee et al. (1993) developed a genus-specific Phytophthora DNA

probe that had no significant cross-reactivity with twelve other Oomycete genera tested. Probes

were also developed for P. capsici, P. cinnamomi, P. megakarya, and P. palmivora, but these

16

were tested for cross-reactivity against only a relatively small number of isolates. Martin (1991)

worked on selection of DNA probes for identification of Pythium oligandrum and P. sylvaticum

and had some success with species-specific identification of the latter. Identification methods

using DNA show promise for more reliable and consistent identification of Pythium and

Phytophthora spp. However, as with ELISA tests for Phytophthora and Pythium spp., more

taxonomic work and development of probes are warranted before they are to be used as a reliable

means of identification and detection of Oomycetes.

BIOLOGY OF PHYTOPHTHORA IN THE AQUATIC ENVIRONMENT

Propagules of Phytophthora. Phytophthora spp. are divided into two categories, which

are delimited by their sexual properties--heterothallic and homothallic. Homothallic species are

capable of sexual reproduction in single mating type cultures, while heterothallic species

generally require two mating types (i.e. A1 and A2) for sexual reproduction. Phytophthora spp.

may also produce asexual propagules, which include zoosporangia, zoospores, and cysts. Some

species also form chlamydospores. The propagule most often associated with the capacity for

multicyclic disease epidemics, however, is the zoospore (Erwin and Ribeiro, 1996).

Thomson and Allen (1974) demonstrated that zoospores were the infective propagules in

leaf baits, because chlamydospores, sporangia, oospores, and cysts were not able to infect

floating leaf tissue in laboratory experiments. In later work they observed chlamydospores,

sporangia, cysts, and germlings to sink without infecting leaf baits while zoospores formed cysts

on leaf pieces within a ten-minute period. Penetration of the leaf tissue and subsequent

zoosporangia formation occurred within 36 hours at 24°C (Thomson and Allen, 1976).

In citrus irrigation water assays, Thomson and Allen (1974) filtered samples through

nylon sieves with pore diameters of 10 µm and 48 µm, and Millipore filters with 8 µm diameter

17

pores. The nylon sieves were used to collect chlamydospores and sporangia and the smaller 8 µm

Millipore filters were used to collect zoospores and cysts. After filtering samples, filters were

inverted on selective P10VP medium and incubated. Further work was performed to identify

Phytophthora spp. isolated. No isolates were obtained when the pore size was greater than 8 µm,

so Thomson and Allen concluded that zoospores were the only propagule present in the irrigation

water.

In another study of recovery of Pythium and Phytophthora from recycled irrigation water,

Pittis and Colhoun (1984) reported recovering a ratio of 1:4 colonies of both genera respective to

the use of 20 µm and 8 µm Millipore filters. This indicated that at least two different types of

propagules existed in the water samples tested. The 8 µm pore probably caught the smaller

zoospores in addition to the larger propagules—chlamydospores, oospores, sporangia—that

would be caught on the 20 µm filter (Pittis and Colhoun, 1984). Charlton and von Broembsen

(2000) demonstrated that over 94% of Phytophthora propagules captured from nursery runoff in

a recycled irrigation system are zoospores.

Survival of Phytophthora. The significance of plant pathogens in horticultural irrigation

systems reflects their ability to adapt to an aquatic environment. The importance of plant

pathogens in any horticultural production system also relates to their ability to survive an

adequate amount of time to be redistributed as potential inoculum. When Shokes and McCarter

(1979) observed the mortality of zoospores in a relatively short time after release into containers,

which were placed in ponds, they postulated that Pythium spp. most likely survive in irrigation

systems as oospores or in infested organic substrates. Phytophthora mycelium and zoospores

survive for up to a few weeks, but chlamydospores of this genus have been observed to survive

up to six years and oospores up to 13 years. Phytophthora spp. are generally considered poor

18

saprophytes (Erwin and Ribeiro, 1996), which at least limits their survival in the absence of a

host. However, the competitive saprophytic ability of P. cryptogea has been reported by

Bumbieris (1979) who considered this species conspecific with P. drechsleri. P. cryptogea has

also been been demonstrated viable in soil after four years without a host (Erwin and Ribeiro,

1996). P. cryptogea has been commonly isolated from water (Ali-Shtayeh and MacDonald,

1991; Bewley and Buddin, 1921; Lauderdale and Jones, 1997; MacDonald et al., 1994; Taylor,

1977; Wilson et al., 1998) and possesses a relatively broad host range.

Thomson and Allen (1976) found that P. parasitica remained viable in irrigation water

for up to 60 days after zoospore introduction. The motile period of these zoospores lasted up to

20 hours, which would allow for spread through an irrigation system. However, when agitation

or physical contact occurs, zoospores rapidly encyst. Turbulence could, therefore, cause

encystment and is possible in an irrigation pumping system (Thomson and Allen, 1976).

Mycelial fragments of P. parasitica were observed to undergo protoplasmic contraction

under conditions of starvation, but remain viable for up to 40 days in non-treated wastewater.

When nutritional amendments were added, they germinated. Other Phytophthora spp. and

Pythium ultimum have been observed to survive in a similar manner (Thomson and Allen, 1976).

Other adaptations for survival have only recently been observed and investigated. When

von Broembsen and Charlton (2000) examined the survivability of encysted zoospores of P.

cinnamomi, P. citricola, P. citrophthora, and P. parasitica in both lake and sterile distilled

water, approximately 80 to 85% loss of viability was observed after 48 hours. However, some

cysts demonstrated repeated zoospore emergence (i.e. production of a secondary zoospore from a

primary zoospore). Zoospore cysts derived from zoosporangia of P. cinnamomi, P. citricola, P.

citrophthora and P. parasitica were observed to germinate and release secondary zoospores at

19

rates of 25 to 73%. These secondary zoospores showed rates of re-emergence from 43 to 62%. A

third generation of an isolate of P. parasitica zoospore cysts showed re-emergence rates of close

to 75%, and observation of a fourth generation occurred. These results indicate methods of

zoospore survival not previously documented and demonstrate another adaptive advantage at

least some Phytophthora spp. possess.

COMMON WATER TREATMENT METHODS

Overview of Water Treatment Technologies. Disinfestation of recycled irrigation

water is often warranted (Buddin and Bewley, 1921, Thomson and Allen, 1976) and many

methods for disinfestation of irrigation water exist. Avoidance of propagules can also be

employed as a means of decreasing the spread of plant pathogens through irrigation systems.

This can be achieved by pumping from a floating inlet pipe to reduce intake of sediments near a

pond’s bottom. Pumping from deep wells could also reduce plant pathogen spread, but is costly

(Shokes and McCarter, 1979).

Filtration is an option for water treatment. Sand filtration has the benefit of maintaining

beneficial organisms in the water, but does not eliminate bacteria or viruses (Wilson et al., 1998).

Micro-filtration is limited by low flow rates and can be used only in highly disease-susceptible,

contained areas, such as propagation (Wilson et al., 1998).

Ultraviolet light is effective unless limited by significant suspended matter and humic

acid, both of which decrease light penetration. In operations where a continuous liquid

fertilization program is used, damage to iron chelate is a potential problem reported with UV

light treatment, and could result in iron chlorosis (Stanghellini et al., 1984). Ozonation has been

reported effective, but is prohibitively expensive (Skimina, 1992). Various forms of chlorine,

such as chlorine gas, sodium hypochlorite, and calcium hypochlorite, have been commonly used

20

as disinfestants (Skimina, 1992). Chlorine, UV light, and ozone all work well, but demand

conscientious management (Wilson et al., 1998).

Liquid chlorine was used as a disinfestation method for the irrigation water at Riverbend

Nursery where water assays in this work were performed. However, in August 2000, a

potentially more effective system that injects chlorine gas into the irrigation water was installed.

Since chlorine is the treatment used at the nursery of interest and is common in many

horticultural operations, a brief overview of the basic principles of chlorine treatment will be

reviewed.

Chlorination. The amount of chlorine put into a water system does not directly translate

into the amount of chlorine actually available. The latter is termed free residual chlorine or free

available chlorine (Daughtry, 1984). Limitation of the amount of chlorine actually available is

due to absorption of chlorine by suspended particles of silt, fertilizer, colloidal particles, and

other organic substances that reduce the efficacy of chlorine treatments (Baker and Matkin,

1978; Daughtry, 1984). Chlorine gas is much cheaper to purchase than liquid chlorine, but many

nurseries are dissuaded from using the gaseous form due to safety considerations (Daughtry,

1984).

It has been demonstrated that 1 ppm of residual chlorine kills zoospores of Phytophthora

cinnamomi after one minute. However, to kill the mycelium of the fungus, 100 ppm for 24 hours

or 200 ppm for four hours was required (Baker and Matkin, 1978). Daughtry (1984) recommends

a level of 0.3 ppm of free available chlorine. The concentration of chlorine that can be safely

applied to plants varies from a lower rate of 2.5 ppm residual chlorine for seedling plants to a

higher rate of 5 ppm for woody or more mature plants.

21

Recent Investigations. Some novel approaches to control of zoosporic fungi have

recently been investigated. Stanghellini and Miller’s (1997) work with biosurfactants as control

agents of pythiaceous fungi in hydroponic systems involved use of certain bacteria, which

produce rhamnolipids that lyse zoospores. Calcium amendments are another novel approach to

control Phytophthora spp. in irrigation systems. Von Broembsen and Deacon (1997)

demonstrated a reduction in the number of viable zoospores released from sporangia with

calcium amendment. Addition of Ca2+ of 20 meq or higher decreased the period of motility of

zoospores. Additionally, quantities of 10 to 20 mM CaCl2 favored germination of encysted

zoospores over production of secondary zoospores. Calcium amendments show promise in cases

where crops are salt-tolerant.

OBJECTIVES

Although recycling irrigation water is increasingly being adopted in Virginia by the

horticultural industry to avoid pollutant discharge, basic research on plant pathogens associated

with recycled irrigation water in Virginia is lacking. The risk of spread of plant pathogens

through irrigation water is of serious concern to Virginia's horticultural industry and, with 25%

of crop agricultural receipts in 2000 contributed by Virginia's greenhouse and nursery industry,

investigations are warranted (Pittman, 2001). Water assays to establish a collection of members

of the Pythiaceae for future work in rapid detection methods and to characterize fluctuations and

locations of Phytophthora and Pythium in a recycled water irrigation system at a container

nursery in Virginia were performed. Additionally, isolates of Phytophthora were identified to

species, and tested for pathogenicity in greenhouse tests to identify those species of most concern

to plant pathologists and horticulturists. Container field plots were also established to assess the

effects of irrigation with naturally infested recycled irrigation water on plants. This research will

22

provide data for development of protocols and recommendations for the industry to best manage

recycled irrigation water and associated plant pathogens.

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