AGRICULTURE, AFLATOXINS AND ASPERGIUllS

PJ. Cotty, P. Bayman. D.S. Egcl and K.S. Elias

Southern Regional Research CenterAgricuhUflll RescllfCh ServiceUnited Stated Department of AgricultureP.O. Box 19687New Orleans. Louisiana 70179

!J'ITRODUCTION

Human activities affect both the size and SD1IClurc of fungal populations.ConsttUCtion. war, recreation, and agricuilure disrupt large expanses of vegellltion and soil;disruption causes redistribution of fungal propagules and makes nutrients available to fungi.Many fungi, including the aspergilJi. exploit these human engineered resources. This re.'iuhsin the association of large fungal populations with various human activities. especiallyagriculture. When crops are grown or animals raised. fungi are also grown. From a humanperspective. most fungi associated with cultivation increase inadvenently. Human activity,bowever. partly dictates which and how many fungi occur and the fungi, both direclly andlhrough fungal products. influence human activities. domestic animals, and even humanslhemselves.

During wann. dry periods. several of the aspergilli increase rapidly in association withcrops. These include aspergilli in the Aspergillus flavur group. Prior to 1960, interest in theA. flavur group resulted both from the use of certain strains in processing of agriculturnJ.products in Europe and the Orient (Beuchat, 1978). and from the ability of some strains toparasitize insects. In the early 1960's fungi in the A. flavur group were implicated as theproducers of aflatOxins (~d.spergillus..D!!vur toxins"), the toxins which poisoned thousands ofpoultry, pigs and trout; in trout these factors were associated with liver cancer (Goldblan and51010ff. 1983). It soon became apparent that aflatoxin! also occurred in the human diet andthat aflatoxins could pass from foed to milk with only slight modification (Goldblaa and510108'. 1983). The most common aflatoxin, aflatoxin B•. was found to be a potent hepalD­careinogen in rats and trout carcinomas were induced at flltes below Ipgkg" body weight(Robens and Richard, 1992). Aflatoxin content of foods and feeds was eventually regulatedin many countries (Stoloff et at.. 1991). In some products. such as milk or infant foods,aflatoxin levels below 0.02 pgkg·· are mandated. Thus, for many, the focus of interest in thisdiverse and important fungal group became the production of aflatoxins.

There clearly are interactions between agriculture, and both aflatoxins and lhe fungiin the A. flavus group. Some consequences of these interactions are obvious. others arevirtually unexplored. The relationship of crop contamination cycles to the life strategies of

noGmar~. EdiIed. by Kcilb A. Powen d '"'~

Pta.aI Pl'es.s. N_ Yoft. 19'J.t

A. flavus group fungi is uncertain. The role agriculture plays in structuring A. flavuspopulations and their tOldgenic potential is also uncenain. This chapter will address someaspects of the intemctions of A. /Iavus with humans and human activities: it includessuggestions on how these interactions may be altered to reduce human exposure to aflaloxinsand other deuimental fungal trail~.

INFLUENCES OF TilE ASPERGILLUS FLA VUS GROUP

Effects of Anatoxins on Humans and Domestic Animals

Although aflatoxins are most often noted for ability 10 induce liver cancer at very lowdoses, they can cause several problems of economic importance during animal production.The presence of relatively high levels of aflatoxins in feeds can lead 10 animal death; rabbits,ducks and swine are particularly susceptible (LD.5&= 0.30, 0.35, and 0.62 mgkg' l

, respectively:Pier, 1992). However, at much lower concentrations. aflatoxins have other effecl~ ondomestic animals including immunosuppression and reduced productivity (Pier. 1992; Robensand Richard. 1992). Once consumed, aflatoxins are also readily converted to aflatoxin M.which occurs in milk and can thus cause both human exposure and sickness in animaloffspring (Pier. 1992; Robens and Richard, 1992).

Incidence or Health Effecls due to Conmminated Foods. In many developedcountries, regulations combined with both an enforcement policy and an abundant food supplycan prevent exposure of human populations, in most cases, 10 significant aflalOxin ingestion(Stoloff et 01.• 1991). However, in countries where either food is insufficient or regulationsare not adequately enforced, routine ingestion of aflatoxins may occur (Hendrickse andMaxwell, 1989; zatba, et a/., 1992). In populations wilh relatively high exposure, a role foraflaloxins as a risk faCtor for primary liver cancer in humans has repeatedly been suggested,but is still not clear (Robens and Richard, 1992). However, aflalOxins cause a variety ofeffects on animal development. lhe immune system and a variety of vital organs. Exposureto aflatoxins, particularly in staples (i.e. com or peanuts) of people dependent upon relativelyfew nutrient sources, must be considered a serious deuiment. The relationship betweenafIatoxins and kwashiorkor may be only one reflection of this detriment (Hendrickse andMaxwell, 1989).

Effects of Aflatoxins on Agricultural Enterprise. Controversies regarding thepossible role of aflatoxins in primary liver cancer of humans are moot in the contemporaryinternational marketplace. Brokers and producers of agricultural commodities have foundaflatoxins increasingly cosily as careful monitoring of aflatoxins limits the use and value ofcontaminated products (Cappuccio. 1989). Regulations in most developed counnies and evenmany less developed counuies restrict the import of contaminated foods and feeds (vanEgmond, 1991: Stoloff et 01., 1991). Assessing the aflatoxin content of crops is a routineaspect of brokering and often a prerequisite of shipping. Contamination is highly variable andallowable concentrations are at such low levels (some below I pgkg·'). that analysis prior toshipping cannot always ensure acceptable levels upon receipt. even if no increases occurduring transit (Horwitz el at.. 1993). This increases commodity costs and can decreasecompetitiveness of imported products. Regulations applied more rigorously to imponed thandomestic products or set at zero, where the limit of detection detennines the enforcementlevel, can serve as barriers to trade which again increase the cost of products. Theseincreased costs may be the primary effect of aflatoxins felt by most consumers in developednations.

Effecls of Anatoxins on Health of Agricultural Workers. Labourers engaged inproduction and processing of commodities may be exposed to aflatoxins through inhalation

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(Shotwell. 1991). Crops grown under conditions favouring aflatoxin contamination oflenbecome covered with large quantities of !t.flavus propagules. Funhenno~,air in areas wherecontaminated crops are produced may contain thousands of propagules per cubic meter (Leett 01., 1986). These propagules. which are mostly conidia, remain associated with the cropsthrough harvest and processing. Conidia contain large quantities of aflatoxins (over 100mg,kg"1 in some strains; Wicklow and Shotwell, 1982). Since most contamination occurs indamaged crop components, fines and dust generated during aop processing have much highertoxin contents the.n the crop as a whole (Lee tl oJ., 1983). The conidia, fines, and dust, maybe inhaled and thus pose an avenue of exposure to aflaloxins; this exposure has beenquantified in certain cases (Shotwell, 1991). Recently, occupational exposure 10 aflatoxinsthrough the handling and processing of contaminated agricultural produCls has been associatedwith increased risk of both primary liver cancer and other cancers (Alavanja tt 01., 1987;Olsen et al., t988).

!tS/Hrgil/us flavus group Fungi as Allergens and Animal Pathogens

Several allergic and infective conditions of humans and certain other vertebrates arecaused by Aspergillus species (Rinaldi, 1983; SL Georgiev, 1992; Wardlaw and Gedes. 1992).These include allergic bronchiopulmonary aspergillosis and invasive pulmonary aspergillosis.The moSI common cause of most of these conditions is Aspergillusjumigalus (Rinaldi. 1983;Sl Georgiev. 1992; Wardlaw and Gedes, 1992). However, other aspergilli, includingmembers of the A. flovUJ group. att: also of[C:n implicated..

Insect Pathogen. During epidemics of aflaloxin contamination. high concenuationsof !to flaViu group propagules are associated with most objects resident in fields. includinginsects; thus insects may serve as vectors (Stephenson and Russell. 1974; Widstrom. 1979).A. flavus readily grows and multiplies on insect damaged crops, insect frass and on inseclsthemselves both as dead debris and as parasitized hosts (Sussman, 1951,1952; Stephensonand Russell, 1974; GOto et 01.• 1988). Many insects typically carry !t.flavus group isolatesinternally and many insects are hosts of at least cenain strains (Slephenson and Russell, 1974;WidslrOm, 1979; Goto et 01.• 1988). Domesticated insects are included among the hostS ofthe A. jlalJUJ group. Domesticated insecl diseases include Stooebrood, a rare disease of thehoney bee which is of minor importance to bee keepers (Gilliam and Vandenberg, t990) andkoji kabi disease of cultivated silkwonn larvae (Ohtomo el oJ., 1975; Geto tt 01., 19118).

Benefits or Aspergillus jI4"us group Fungi

Industry. Fungi in this group have had a long history in processing to inctt:aseproduct utilily and value. A. flavus group strains are used 10 produce enzymes for foodprocessing and other industrial uses and even to produce therupeutic products such as uruteoxidase and lacloferrin (Chavalet et of., 1992; van den Hondel et at.. 1992; Ward et al.,1992). A variety of tntditional fermented food prodUCl<; have been made with fungi in the A.flavus group for centuries (Beuchal, 1978).

Ecological Benefits. Although A. flal'us group fungi are OOt commonly recognisedas beneficial. these ubiquitous organisms become dominanl members of the microflora undercertain circumstances and exen multiple influences on both biota and environmenL Thesefungi are important degraders of crop debris and may play roles in solubilising and recyclingcrop and soil nutrients (Ashworth tl 01.• 1969; Griffm and Garren. 1976). !t.j1avus can evendegrade lignin (Bens and Dan. 1989). As insect pathogens. these fungi may serve 10 limitpest populations (Wadhwani and Srivastava. 1985) and have even been considered potentialagents 10 replace chemical insecticides (RobertS and Vendol, 1971).

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Contamination Cycles

Contaminated components. A. flavw causes a variety of planl diseases typical oflargely saprotrOphic "weak" planl pathogens (Widstrorn, 1992). These diseases include boll,ear, and pod rots which result in both decreased yield and reduced quality (Shurtleff. 1980;Watkins. 1981). However. crop infeclion by A.flavus takes on a different importance thaninfections for which concern might focus on yield and quaJity loss, or increased free fanyacids. Aflatoxins are compounds regulated in pans per billion; yet, these toxins occur incertain infections at COncenb"8.Dons over 100.000 ug.kg-'. This situation causes high-toxin­containing components to greatly exceed in cost the vaJuc of the same components if notcontaminated_ Variability among componentS ofcrops in aflatoxin conlent is extreme (FigureI). Most infected components contain low aflatoxin concentrations (below SO pgkg·').However, a small percent contain very high toxin levels. at times exceeding 500.000 ~gkg"l

(Cucullu el at.. 1966; Schade er of., 1975; Lee et of.• 1990; Steiner er of., 1992), In manycases, elimination of highly contaminated components (over 1.000 lJg.kg·') would resuh in acommodity with an acceptable average anatoxin conlenl (Schade et af.. 1915; Steiner er at.•1992).

Crop components damaged by wounding or severe stress are colonised and decayedby a variety of fungi During hot and dry conditions. fungi in the A. flovus group outcompete many colonising microbes and become lhe prominenl fungi degradin8 damagedcomponents. In most crops lhe majorilY of contamination occurs in damaged plant pans(Wilson et al.. 1911; Lee et. aL, 1983; COllY and Lee, 1989). Damaged seed can be sortedfrom high value crops for less profitable use such as production of yegetable oil. However,crushing contaminated seed to produce oB concentrales analOxins in the resulting meal whichis used for feed Such toxic meal caused the first recognised anatoxin problems; peanut mealcaused turkey X disease in England and couonseed meal caused rroUt hepatoearcinoma in lheUnited States (Goldblatt and Stoloff, 1983). Such meal must either be detoxified (i.e. throughammoniation) or put to non-feed use (Park cl al.• 1988).

Geo&raphy determines frequenc.y and severily. Geographic location greatlyinfluences frequency of contamination_ Many agricultural areas at low eleyation and betweenlhe latitudes 35 N and 35 S have perennial risk of contamination. Countries in this zone(whic.h include many countries with insufficient food supply) may view elimination ofaflatoxins from lhe food supply differently than countries whose major agricultural lands lieOUI of this zone (Le. developed countries in Europe and Nonh America). Producers ofcontaminated prodUCIS may base allowable levels of aflatoxins on toxicological data. whereasconsumer nations which rarely produce contaminated products may base allowable levels atthe lowest level detectable (Stolofr et aJ.• 1991).

Contamination cycles can be considered perennial. sporadic or infrequent ba.~ onlocale and crop. In aU three situations, populations of A. flavw are long tenn residents.However. populations in different areas differ in magnitude (Figure 2) (Griffen and Garren.1914; Manabe et al.• 1978; Shearer Cl 01., 1992) and possibly in the distribution of bothqualitative and quantitative trailS (Manabe el af., 1978; Cotty. 1992b). During periods notconducive to contamination, perennial areas (i.e. the desen valleys of Arizona; Lee et al.•1986) suppon higher A. flavus populations than areas with infrequenl contamination, i.e.midwest com producing areas (SheaIel" et al.• 1992). Areas with sporadic contamination mayhave perennial contamination at low leyels but. have less regular exposure to imponantpredisposing factors such as hot, dry conditions. i.e. contamination of com in certain areasof the southeaslern United States (Widstrom. 1992) or insecl pressure. i.e. pink boUwonnpressure on cotton in western Arizona (Cony and Lee, 1989). During peri<Kls conducive tocontamination. a shift in the micronora occurs and aflatoxin producing fungi becomedominant colonisers and decayen.

•

Processes through which crops become contaminated with afiaLOxins are varied andcomplex (Diener et al., 1987). However. certain generalities might be suggested.Coo.tamination cycles may be divided into three phases: Prebloom, Crop Development, andPost Maturation (Figure 2).

Prebloom. Contamination does nOl occur in the field during the period after cropremo at and prior to bloom. However, both me micro6ora and crop may becomep~sedLO contamination. During this phase: 1. propaguIe (conidia. sclerotia., colonised organicmatter) are dis~ed through cultivation. planting, pruning or omer activities of animals(including man) or the environment; 2. A. flavus populations fluctuate, fil'St decreasing aftercrop removal and then, if conditions are t: ourable. increasing on debris from current and

0- 0.1-2.5 glkg

- 2.5-15 Q/kg

• = CMlf 5 glkg

0- &0010'

Vi urt L DiSln'bution and afbioxin c tent or maize mcls on an ear (drawn from Ul in Lee tloJ.• 19&0)and bolls on a plant (Colty and lee. 1990). Conlamimnion is highly variable and nOl all infeeted seed becomescontamirulled.

prior crops (Ashworth et al., 1969; Griffen and Garren, )974; Lee et 01., 1986) 3. The cropmay become predisposed by long periods of drought or by luxuriaDl growth followed bydrought. (Cole et al., 1982; Deiner er al., 1987; Shearer el al., 1992); 4. Overwinteringinsects emerge and develop.

rop DeveJopmenL From nowering LO maturation seeds and fruiL~ are vulnerableto variou perturbations. During this phase: I. If conditions are hot and dry, populations ofthe A. /lavus group, in canopy and soil, will outcompete many s prophytic microbes andincrease iDiu. 2. High tempcra~ and/or drought IreS may interfere with cropdevelopment and weaken plant defence making the crop more susceptible to infection andcontamination (Jones et al., 1981: Cole et al.• 1985; Wotton and Strange. 1987; Widstrom,

5

1992). 3. Wounding of fruits at middle to late tages of development can lead to portionsof the crop wilh vel}' high toxin level (Lillehoj et aI., 19 7; Cony, 1989b). In seveIa1 cropmost aflatoxin is formed during this phase and in certain locations crop predispo aJ tocontamination can be attributed to specific wound types caused by specific insects. Examplesare pink bollworm exit holes in cotton in the desen valleys of the western United States(Cotty and Lee. 1989). maize weevil damage in the southern United States (McMillian et al.•1987). navel orange worm damage in nuts in the western United States (Schade et al.• 1975;Sommer et aI., 1986), and lesser com stalk: borer damage in peanu in the southern UnitedStaleS (Lynch and WIlson, 1991). 10 me crops. components prevented from mamring dueto suess or early harvest are particuJarly vulnerable to contamination (Cole et al., 1985;Lynch and Wilson, 1991).

EnvIrOllm~t determ e. tile nItUde of A.~rglllu. navu. popul8tlonll,-. • Location 1 ... _. II ~on 2 ••••• locatlon.s

""""t operIIIloM "lIlY~ clM\iIge end

pte It,pMe the ciop

."

Year 2

,.. .;

<i.I

Figure %. ConlamiDation cycles can be divided inlo lhree pIWes. LocaJ conditions dctennir.e bOth theextent of conraminalioo and the magnitude or A. flavlls populad iated with lhe crop. BoJ:cdinformation appl' to all crops.

e

Post Maturation. Most crops are susceptible to aflatoxin contamination at maturityand if the crop was grown in an area with perennial contamination or during a periodconducive to contamination, the mature crop will be associated with large quantities of A.f1avus group propagules. These propagules remain associated with the crop as it awaits harvestin the field, during harvest, field storage (i.e. peanuts in windrows. couon in modules),shipment and processing, and even during storage by the end user. Exposure of the maturecrop to periods of wetting and drying under warm conditions may lead to increasedcontamination. Aflatoxin concentrations are known to be dependent on environmentalconditions and competing microflord (see Strain Isolation and Accumulation of Aflatoxins).Mature fruits and seeds are living organisms and factors which compromise seed health, suchas wounding or stress, predispose these products to infection and contamination. Harvestoperations can simultaneously damage crops and introduce A.flavus into wounds (Schroederand Storey. 1976; Sommer et a/., 1986; Siriacha et a/., 1989). Insect activity after harvestcan disperse aflatoxin-producing fungi and, by increasing host susceptibility, increase aflatoxinlevels in a manner similar to insect damage during crop development (Dunkel, 1988). Thesame insect can affect contamination both prior to and after maturation (ie. the navel orangeworm on pistachios).

Post maturation contamination dictates that each handler of the crop be responsible andminimize the potential for aflatoxin increases. Thus dairies which purchase feed withundetectable toxin must still store the feed properly or contaminated milk may occur. Withindeterminate crops (e.g. cotlon) crop development and post maturation phases may occursimultaneously and with all crops the prebloom and post maturation phases occursimultaneously, although at different locations.

Initially, the crop development phase was ignored because all contamination wasthought to occur post harvest; recently. most research has been directed at contamination

1.0

~~ 0.9CD

::l>. 0.8

~ Ea: 01:s .= 0.]

~ ~(i) 0.6

.. ' '.4: ii~~,js··.A. parasltlcus A. nom/us:. ,.u~J',t:~~~.:"""TIPU ISOLAna mt! 8~':f

'"

S straine ISOLAtES

Lstrain15 f5OVotti

Figure J. Phenograms of A.f1avlIs group isola.tes. Taka-a.mylase data from Egel and Cotty. (1992); RAPD datafrom Bayman and Cotty, (1993).

7

before harvest, sometimes without distinguishing periods of active crop development fromperiods after maturation (UlJehoj er al.• 1976; Goldblatt and Stoloff. 1983). Thecontamination process can be divided many different ways besides those presented here.However. failure to segregate the contamination process into different phases may result indata that suggests no clear pattern and apparent contradictions.. For example., in Arizona. mostcouonsecd contamination occurs during crop development in cononboUs damaged by pinkbollworms in the absence of rain (Cony and Lee, 1989). Still. rain on a mature crop awaitingharvest can lead to significant contamination during post maturation, even if developing boDswere not damaged (Cotty. 1991). Similarly, a great fervour occurred about contamination ofthe midwest U.S. com crop, in the field, during droughts of 1983 and 1988 (Kilman. 1989;Schmitt and Hurburgh, 1989; Shearer el at., 1992). Yet, in Thailand, contamination typicallyoccurs during the wet season, not during the dry season (Goto et at., 1986). In Thailand'srainy season. contamination occurs during poSt maturation (Siriacha et aI, 1989); in themidwestern United States. il typically occurs during crop development (Lillehoj er al., 1976).

FUNGAL POPULATIONS

Diversity

Species of AIIatoxin-Producing Fungi. There have been a variety of taxonomicschemes used to classify A.flavus group strains (Thom and Raper, 1945; Klich and Pilt, 1988;Samson and Frisvad, 1990). Each species represents an assortrTlent of strains which behaveas clonal organisms with the exception of occasional parasexuality between members of thesame vegetative compatibility group (papa, 1984, 1986). For the purposes of this discussionwe will place aU isolates within this group into fout species A.flayus, Aspergillus parasiticus,A. f1omjus, and Aspergillus tamarjj. Depending on interpretation, these species are supponedby clustering algorithms based on DNA polymorphisms (Kurtzman er al.. 1987; Moody andTyler. J99Oa,b; Egel and Cotty, 1992; Bayman and Cony, 1993). A. tamarii is of minorinterest here because no isolates in this species produce aflatoxins. A. lamar;; isolatesappmntly have some marlcedly different adaptations than the remainder of the group and A.lamarij is more distantly related to the other three species, than the three are to each other(Kurtunan el al., 1987; Klich and Pitt, 1988). Aspergillus oryzae and Aspergillus sojae 81e

apparently derived fromA.flavus and A. paraJjlicus. respectively (Kurtzman el al.• 1986) andwill be mentioned only in an industrial context A. nomius was named after the genus ofalkali bees from which several isolates were obl3.ined (Kunzman et at.. 1987). A. ncmiuscomprises a group of strains that are distinct by both physiologic and molecular criteria(Kunzman el al.. 1987; Bayman and Cony, 1993). The name "nomius" may be misleadingin associating this species predominantly with the alkali bee when isolates are known fromseveral crops. including wheat (the type isolate) and peanuts (Hessel Line el at., 1970).

Diversity Wilhin AsptrgiOu$ flavus. Within each of the three aflatoltin producingspecies. there is 8 great deal of variability among isolates. It may be, that if we sought outall the unusual or atypical isolates within this group and examined them, we would find acontinuum as suggested by Thorn and Raper (1945). Indeed. based on polymorphisms in theTaka-amylase gene, we have found strains intermediate between A.flavus and A. parasilicusas well as A. ncmius isolates almost as different from the A. nomius type strain as the A.parasiticus type from the A. flavus type (Egel and Cotty, 1992; see Brazil nUl isolate inFigure 3). Variation among isolates is evident in genetic, physiological and morphologicalcharacters. Each of the above species is composed of at least several VegetativeCompatibility Groups (YCGs) and A. flavus is composed of many (Papa, 1986; Bayman andCony, 1991; P.J. Cotty, unpublished). Physiological and morphological traitS are typicallymuch more consistent within a vea than within the spe<:ies as a whole (Bayman and Cony.

8

1993). Thus, a large portion of the variability perceived within A.flavus reneclS divergenceamong VOOs. 11ris divergence has resulted in consistent differences among VCGs in severalcharacters. including enzyme production. plant virulence, sclerotia! morpbology. and otherphysiological trailS (Cony. 19890; Cony el al.• 199Ob; Bayman and Cony. 1993).

.... ... • •, •/ • •\ - , B .- ••/ ~

.. ·., / .A - C -·

•I • • •- •

•~ •- • . •-- •,

I • • •0 E

fiprt •. Silhouettes of scJetolia produced by aflalOxin producing isolalcs durina 30 days growIh al 32 "Con Cupek's agar. A is an unusual isolalf: or A. ncmillS (rom a Bruil nul; B b an L strain isolate of A.jfavuJ; C is an S s!rain ilOlale of A. /lavllJ: 0 is lhe type isolate or A. nomillS. E i5 an isolalc of A.pora.tltfclU. All bars are J mm.

Variability in production of aflatoxins. especially among It.flQllus isolates. has oftenbeen reported and discussed (Joffe. 1969; Davis and Deiner. 1983; Oevstrom and Ljunggren.1985). It.flavus isolates may produce anywhere from no detectable afl810xins «I pgkg.l) toover 1.0000,OOO pgkg.l. A. parasiticus and A. ncmius produce B and G aflatoxins, and A.porasiticus produces aflatoxins far more consistently than It.flavus (Hesseltine el al.• 1910;Domer et al.• 1984; Kunzman et af.• 1987). Too few isolates of A. nomius have beenexamined to discem consistency. A. flavus is generally considered to produce only 8aflatoxins (Samson and Frisvad, 1990); however. this observation is dependent on how thedefinition of A.flavus is n:stricted (Saito el 01.• 1986; Klicb and Pill. 1988). Taxonomy aside.variability in toxin production. and other strain differences indicate divergence and possiblediffen:ntia! adaplation. This variability can be a tool {or disceming functions of variable trdilS(Cleveland and Colly. 1991); il may further be used to develop a better understanding of theecological niches to which sltains are adapted.

On the basis of physiological and molJlhological criteria, A. flavus can be divided into IWO

strllins. S and L (Cony. 19890). Isolates in the S strain of A.flavus (actuaUy a collection ofstrains which belong to numerous VCGs; Cotty. 19890; Bayman and Cotty. 1993; Cony et01.• 199Ob) produce numerous small sclerotia and fewet conidia than other A.f1a'tlus isolates:(COlly. 19890: Saito et al.• 1986). The L strain is composed of lhe so called "typicalM isolatesof A. flavus (Saito et 01.• 1986) which produce larger and fewer sclerotia. Some keydifferences between the Sand L strains are outlined in Table I.

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Table 1. Key characteristics of the S and L strains of Aspergillus jlavus

Character L strain S strain Reference'

Sc: urn sizeProduclioo of aflatoxinsProduction of conidiaProduction of sc.1cmtiaon potalO de.11rose agaron S% V~ juiceVirulence to cottonPcctimse producI.ionPrimary habitat

Average> 300 mmVariable. Zero 10 HighHeavy

one 10 many. )()cm·J

one \0 few, < lem".HighConsis\C1lIAerial?

Average< m A. BC . IenL High A.., BLighl A.., B

Many > SOcIJf' AMany IOcm-~ 10 SOcm·l Ato 10 High AVmUble CSoil?

IReferences: A = COlly. 19890: B = Sairo tf 01.• 1986: C = COlly tl al.. 1990b

Imporlance of Infrequent trains to ontamination. The etiology of aflatoxincontamination, and the relationship of both the size and structure of Aspergillus populationsto contamination is complicated by the importance of unusual strain types whjch occur at lowfrequency. Aflatoxin contamination is a peculiar and frustrating agricultural problem becauseless than 1% of the crop may be contaminated with levels high enough to make the averageof the entire crop exceed allowable concentrations (Figure 1). During attribution of cause.infrequent but highly toxigenic strains may easily be overlooked or not identified as potentialaflatoxin producers. Such may be the case with isolates belonging to the S strain of A.flavus. Due to colony and scLerotiaJ appearance (Figure 4) S strain isolates may be passedover in favour of co-occurring "typicaJ" or L strain isolates. SeveraJ vi itors to ourlaboratory have been urpri ed at the identity of S strain isolate and have returned home to

discover the occurrence of S strain isolate at their locale. In soils of several areas of thesouthern United States. the S train incidence average around 30% (Cotty. I992b). Onaverage S strain isolates produce much higher aflatoxin levels than L stnlin isolates, and alsomore sclerotia and fewer conidia (Saito el 01., 1986; Cotty. 19890) (Table 1). Predominanceof conidia of L strain isolates on mature crops may at times iorerfere with attribution ofconta.mination to S strain isolate actually inciting the problem.

Another relatively infrequent aflatoxin-producing fungu i ·A. nom;us. A. nom;usisolates can produce large quantities of aflatoxin but may be misidentified as A. parasilicuswhich produce' the same aflatoxin (both B and G) and roughened conidia (Hesseltine et al.•1970; Kurtzman et af., 1987). A case in point i an unusual A. 110m/us isolate from a store­bought brazil nut which comained 8,400 ...gkg"' tow aflatoxins (Figure 4). This isolateproduces large quantities of aflatoxins and. based on polymorphisms in the taka-amylase gene,differs almost as much from other A. /lomius isolates as A. paras/ticus differs from A.flavus(Egel and Cotty, 1992) (Figure 3). This isolate is clearly unusual, but it incited significantcontamination in the marketplace. urthermore, such rare highly contaminated nuts are theprimary source of contamination in brazil nuts (Steiner el al., )992).

Diversity in EcologicaJ Nich • Fungi in the A. flavus group are broadly adapted toexploit many organic nutrients and to infect a variety of animal and plant hosts. StrainsmuSt adapt to compete in ecological niches which provide long term urvival. Many trainswith diverse adaptations clearly have orne ucces in exploiting crop related resources.Ho ever, other nkhes which may only upport small fungal populations relative to crop

sociated niches. may have been occupied 0 er long periods by cenain trains. Differencesamong these "minor" niches may drive strain diversification. imiJarly,mbility of minor

10

niches may stabilise the character of minor strain types. Relatively stable minor niches mayhave greater long Ieml imponance than vast crop resources, because suitability and quantityof crop related resources oscillate widely in response to the environment. insect herbivory,changes in agronomic practice and the crop itself.

Wick.low (1982) showed lhal strains of lhc A. flavus group used as Koji moulds(moulds used 10 produce fennented foods) (Beuchat, 1978) genninate faster and have largerspotts than wild strains of the species from which these moulds were probably domesticated;thus during domestication. the Koji moulds might have developed traits which favour rapidnutrient capture (and success during intra.'ipeciflC competition) and lost traits which are notadaptive in the Koji environment. i.e_ aflatoxin-producing ability (Wicldow, 1982). DNArelatedness among strains of A. flavus and A. parasiticus and their Koji mould equivalents.A. oryzae and A. sojae, suggest that the Koji moulds were indeed derived from the wildspecies (Kurtzman et 01.,1986: Egel and Cotty. 1992). However, anributing adaptive valueto Koji trait~ is speculative in the absence of experimental dall!.. Similarly, strain variabilitysuggests multiple adaptations, but our assignment of specific functions to adaptations islargely speculative.

Strains of the A. flavus group may not only differ in host or nutrient use, but also inhostJnuDient location and Strategy to exploit resources.. Members of this group are verycommon both in and above the soil. Although all A.jIavus group strains contribute to the soilbiota. certain strains may be better adapted to capture resources above the soil. Smallsclerotia and reduced sporulation among S strain isolates may imply adaptation to infect andClpturC resources in the soil whereas relatively large sclerotia oflen facilitates aerial infectionand nutrient capt~ (Garrett, 1960). S strain isolates may have diverged from other A.flavusstrains through adaptations to the soil environment. There has been a general assumption thatsclerotia of this group serve primarily to produce conidia after non-<:onducive periods(WickJow and Donahue. 1984). Sclerotia of other fungi can genninate directly to infecthosts or capture resources (Coley-Smith and Cooke. 1971); this may also be an imponantrole for sclerotia in the A. jIavus group. panicularly for S strain isolates which appear todisperse via numerous. small sclerotia.

A. parasiticus has also been associated with the soil environment (Davis and Diener.1983) because in certain locations (e.g. Georgia), it occurs more frequently on peanut.'l thanon com (Hill et oJ., 1985). Although relatively few isolates have been compared. in moStcase.s, A. flavus isolates are more invasive of crop tissues, even peanut tissues. than A.parasiticus (Calvert et aJ.• 1978; Zummo and ScOlt. 1990; Pin et aJ.• 1991). Furthermore,based on OCCUJTenCe of G afIatoxins (produced by A. parasiticUS and A.. nomius, but not A.JIm'us) A.flavus produces most contamination in peanuts (Hill et aJ.• 1985; Maeda, 1990).Conflicting observations on the association of A. parasiticus with peanuts may reflect fungaladaptations 10 soils or conditions in certain locales where peanuts are a major crop and lackof adaptation to other locales. This is supported by a low frequency of A. parasiticus insevent.1 agricultural areas and failure of inuoduced A. parasitcus strains to overwinterefficiently at certain locations (Davis and Diener, 1983; Zummo and Scott, 1990: Cony.1mb).

A..flavus strains produce large quantities of extracellular enzymes (van den Hondeltt al., 1992) which probably enhance their ability to utilise a broad assortment of organicresoun:es. Enzyme polymorphisms have been used to suggest a role for specific enzymes(elastase and pectinase) in fungal virulence. Certain stnlins of A.J1avus have reduced abilityto rot cotton bolls and spread between cotton boll locules (Cotty 1989b). This reduced abilityis associated with failure to produce a specific pectinase isoenzyme. P2C. both in cuh~ anddeveloping cotlonbolls (Cleveland and Cotty. 1991; Brown et aJ.• 1992). tn one populationstudy. 5()tI, of S strain isolates and no L suain isolates failed to produce P2C. Thispolymorphism suggests cenain S strain clones are not dependent on efficient colonisation ofplant hosts. S strain isolates might primarily exploit soil debris and/or insect hosts and thus

11

not require high pectinase, Even though not optimally adapted to exploit plants. pectinaseP2C deficient strains do occur on the commercial crop and can cause significantcontamination (Cotty, 1989a: Cotty et ai.. 1990b). Therefore, specific adaptalion to a cropis not required for strain conuibution to contamination. Analogous to P2C variability isvariability in production of elastase (an alkaline protease). All A.flavus strains isolated frompatients suffering from invasive aspergillosis produced e1ast.ase whereas strains from otherorigins produced elastase Jess frequently (Rhodes et ai., 1988). Thus. a role for elastase inhuman pathogenesis has been suggested (Rhodes et ai., 1988), although this role is stillcontroversial (Denning et ai.. 1992). A. flavlL~ is an opportunistic human pathogen and it'sunlikely that A. flavus elastase evolved to pennit infection of mammals. The ecologicalfunction of elastase is nol clear, however, elastase production may be directed at exploitationof dead mammals or insects (Charnley, 1989; Malanthi and Chakrabony, 1991).

A.flavus has an intimate relationship with insects. particularly lepidopterans (Sussman.1951, 1952). Excretion of large quantities of diverse enzymes, a characteristic of the A.flavus group (van den Hondel et 01.,1992), may facilitate mutualism as well as parasitism andsaprophytism (Martin, 1992). Insect use of fungal excreted enzymes that degrJ.de or detoxifyplant products can drive development of fungal-insect mutualisms (Martin, 1992). The A.flavus elastase actively degrades multiple enzymes in alkaline environments (Rhodes et 01.,1990) and is relatively stable among other proteases (van den Hondel et ai., 1992; PJ. Cottyand lE. Mellon, unpublished). Such activities might ameliorJ.le the lepidopteran gutenvironment (ie. alkaline and high protease activity) (Martin, 1992) and permit strainestablishment and retention. Similarly. aflatoxins may exen influence on insect immunesystems (Charnley. 1989) pennitting fungal str.un retention. A. f1avus-insect relations meetseveral predictions of mUlUalistic relations including fungal asexuality and lack of specificity(Martin. 1992). However, production of a potent insecticide and/or other virulence factors(Sussman, 1952; Ohtomo et ai .• 1975; Drummond and Pinnock, 1990) within host tissuespreclude full mutualism and allows a shift from avirulence to virulence. The associated hostdeath may benefit both saprophytic insect exploitation and movement to plant resources(Bennett, 1981). Speculations about the nature of the relationship aside, diverse anhropodsvector A. flay/IS group fungi, predispose crops to aflatoxin contamination and serve as bothhosts and predators of many A. j1ayus group strains (Widstrom, 1979). In the latter two rolesthese animals may exen strong selective pressure on fungal strain character and the fungi mayexert considerable pressure on insects (Rodriguez et al.• 1979; Wadhwani and Srivastave,1985).

Specialisation of strains seems not to include pathogen-host specificity, or at leastspecificity has not been shown. Sussman (1951) showed diverse lepidopterans were infectedby the same strain of A. flavus and isolates from one crop typically can infect andcontaminate other distantly related crops (Schroeder and Hein, 1967; Brown et ai.. 1991).Similarly. the life strategies of strains causing aspergillosis in poultry and humans are clearlynot directed al specifically exploiting those hosts. Different crop associations of A.parasiticus and A.flavus strains may reflect either as yet undescribed adaptations to specifichosts or other differences in ecological adaptation and life strategy (Moss, 1991) (see above).Many adaptations in this group relate to aggressive saprophytism at elevated temperature andunder relatively dry conditions. As pathogens, these fungi genemlly exploit wounded orstressed hosts and avoid taking on host defenses directly, although A.flayus does elicit plantdefense mechanisms (e.g. enzyme production and phytoalexins) (Mellon, 1991.1992). Still,healthy and non-compromised hosts (both plants and animals) can be infected (Barbesgaardet ai.. 1992; Pitt et at., 1992). Infection of healthy plant parts in the absence of symptomsmay occur regularly, even if these infections do not include invasion of living host cells.Indeed, through serial isolations. systemic plant infections by A. flay/IS group strains havebeen observed in com. peanut and CotlOn (Klich et ai.. 1984; Pitt et al., 1991; Mycock er aJ.,1992).

12

Innuences or Agriculture on Fungal Populations

Fungal Population Structure. Populations of A.flavus group fungi are complex. AUspecies wilhin the group may occur on the same crop or in the same field (Schroeder andBaUer, 1973; Davis and Diener, 1983; Cotty. 1992b). The greatest infonnation on populationstructure is available for A.flavus which is composed of numerous vegetative compatibilitygroups (Papa. 1986; Bayman and Cotty, 1991) (Figure 5). Populations are complex at everylevel with multiple strains occupying gram quantities of soil and individual crop pieces. YCGcomposition of the population infecting a crop does not nece.uarily reflect the veocomposition of the population within the soil in which the crop is grown (Colly, 1992b).Furthermore, during crop production new resources for A. flavus to exploit become availableand population composition may change very rapidly (Bayman and Cotty, 1991; Cotty, 1991b.1mb); apparently these fluxes in population composition are driven by establishment ofrelatively ntre VCOs on newly available te.'iourccs. There is little infonnation on A. fIavusgroup populations in the absence of agriculture and little infonnation on fungal communityresponses to agricultur.tl methods (Zak, 1992). However. it is clear that cultivation disturbsand homogenises the soil environmem in which these fungi reside and in so doing mustdisperse conidia, sclerotia and colonised organic matter. At the same time both cultivationand aop development create immense resources for fungi to use. Although disturbancegenerally ~hs in decrea.o;ed species richness and heterogeneity, (his sudden abundance ofresources during environments favouring the A.fIavus group may pennit noncompetitive SIrllincoeJtimnce (Zak. 1992) and a temporary increase in the diversity of strains exploitingpanicular resources.

Selection or Fungal Strains. The A. flavus group is broadly distributed but, in theabsence of crop cycles. A. flavus group populations are generally maimained at relatively lowlevels (Angle er ai., 1982; Shearer et af., 1992) and in the absence of a conduciveenvironment, A. fIavus populations also maintain low levels on crop resources (Griffin andGarren, 1974; Shearer et a/., 1992). Thus. during conducive periods, there is a potenlial forcrops to exen tremendous influence on strain growth and selection. Slrains infecting crops arediverse in many characters including type and number of sclerotia. toxin producing ability,veo. and even virulence to plants. This diversity among infecting strains suggestS thatagriculture does not aggressively select specifIC fungal types. However, a Jack ofrequ~mentfor aflatoxin production during crop infection and during fungal increases on crops (Cotty,19890) may permit disproponionate increases in aJoxigenic strains (Bilgrami and Sinha,1992). Furthermore. the imponance of aerial dispersal to spread through a crop may causethe high sporulating L strain of A.flavus to outcompete the low sporulating S strain duringsecondary spread in the canopy. An as yet unknown specific strain-vector association couldalso pennit strain advantage. Cropping process. crop types. geography. and/or climate mayselect certain slrain types (Shroeder and Boller, 1973; Lafont and LafORt, 1977; Wicklow andCole. J982; Shearer er a/., 1992). However, muhiple-year experimentS with more rigorousdesign are needed to reliably establish such selection. if present Studies should also utilisestrain identification methods that are more specific lhan ability to produce either toxins orsclerotia. Vegetative compatibility analysis has been shown to be useful for monitoring thebehaviour of specific strains over both time and space (Cotty. 1991b. 1992€) and we recentlyfound differences among cotton producing areas in the proponion of A. j1lJ\lUS L'iOlatesbelonging to the S strain (Cotty. 1992b).

Crops might exert different influences on populations by exposing strains to eitherdifferent substrates or resistance factors. Crop components for which contamination is aconcern (i.e. nuts or kernels) do not exe" the only nor often the major influence onpopulations; other parts (l.t. leaves, slCmS, floral parts. cobs) may playa greater role informing and maintaining the overaU population (Zummo and SCOtt, 1990; Kumar and Mishra,

13

1991). Perennial crops (i.e. tree nuts) may maintain and select strains on perennial parts andlong season crops (i.e. cotton) may provide longer periods of increase and shoner periodsbetween crops than shon season crops (i.~. com). The nature and magnitude of plant debrisand it's successful survival between croppings may be an imponant detenninant of populationsaucrure and magnitude (Jones, 1979; Zummo and SCOtt, 1990). A.flavw; can colonise verylarge proponions of plant debris associated with crops and this debris can yield largequantities of conidia (Ashworth ~t 01., 1%9; Stephenson and Russell, 1974). Variation amongcrops in insect microfiora may also influence the composition of fungal populations. Thisphenomenon might occur due 10 differences in herbivory or variabilily Ilmong insect hoslSin both life cycle and susceptibility to fungal saains.

vca..••~~••'"a.HOI.J •••L 0••

Fip~ 5. Diagl1Jl'll1\aUc represctlt:llion of the veget3live compatibility group (VCG) composition DC Isingle 3ificultur:&! field. Marter rrequency indicalCs the lelalive incidence cllhe Ie~lcd vee wilhinthe fJdd. M:ner shape does not reflect morphologjcal dirrerences among VCGs. Only VCGs thai make liPg.rea1Cf than two percent or the populalion are lepresenlcd (Bayman and COllY. 1991).

Adaptive Value and Ecological Significance or Anatonns

Through their effects on agriculture. afIatoxins prove to be ~non-nuttitional chemicalscontrolling the biology of other species in the environment." Torssell's (1983) definition orsecondary metabolites. A characteristic of secondary metabolites is a principally unknownfunction (forssell. 1983), however. consideration of the adaptive value of aflatoxin! isappropriate when discussing interactions among aflatoxins. Aspergillus. and agriculture.

Protection or Survival Structures. High concentrations (3 mgkg' l to 132 mgkg"'aflatoxin 8.) of aflatoxins occur in both conidia and sclerotia of aflalOxin·producing strains(Wicklow and Cole. 1982; Wicklow and Shotwell. 1982; Cony. 1988). The presence ofafIatoxins in sclerotia has received the most attention because sclerotia are long tenn survivalstruCtures and aflatoxins are highly toxic to a variety or predators of rungi, espec;:iaUy insectS(Wright et 01.. 1982: Willetts and Bullock. 1992). Sclerotia of A..flavus group fungi typicallycontain an extenSive array of other toxic metabolites in addition to afIatoxins (Wicklow,1990). Some of these metabolites are not found in other fungal structures and in combinationwith afIatoxins these toxins may form an elaborate chemica! defense system directed at

14

protecting sclerotia from insect predation (Wicldow. 1990; Dowd. 1992). Indeed kojic acid,• metabolite of most A.flavus strains. can synergistically increase the toxicity of aflatoxin 8 1

In catcpillan (Dow<!. 1988).Long term survival for A.flavus group propagulcs requires resistance to degradation

by microorganisms during conditions (wet amVor cool) nOt conducive to successfulcompetilion by A.flavw. Bacteria are active under these conditions and, although aflatoxinsarc not very inhibitory to fungi, they do inhibit many bacteria at concenaations present insclerotia (Burmeister and Hesseltine, 1966. Arai et al.• 1967. Angle and Wagner. 1981).Aflatoxins even inhibit cenain known bacterial antagonists of A.flavus (Kimulll and Hirano.1988). However, pure aflatoxin BI is rapidly degraded in diverse soils (Angle, 1986), andthus to have long term effects, afIatoxins may themsclvc.~ have to be shielded fromdecomposition. a condition possibly provided by the sclerotial rind (Willens and Bullock.1992).

A.flavus strains which produce sclerotia may either produce aflatoxins or not (Bennetttt aI.• 1979; Cotty. 1989a). Funhennore. sclerotia of the same strain with differing aflatoxincontent can be produced by growing sclerotia on different substrates (Cotty, 1988). Sclerotia.from multiple sources. with different aflaloxin contents could be evaluated for longevity infield soil. resistance to microbial degradation. and insect predation. If aflatoxins contributeto the defense of sclerotia. some level of correlation between aflatoxin content and sclerotiumresistance should occur.

Association with Sclerotia. A. relationship between sclerotia and aflatoxins has beenrepeatedly suggested (Mehan and Chohan. 1973; Sanchis et al.• 1984). This is not astraightforward relationship because. ira vitro. certain fungal strains produce aflatoxins but notsclerotia and vice versa (Bennett et al.• 1979). The situation is further complicated byanributing quantitative differences in toxin producing ability 10 the tendency of a strain toproduce sclerotia (Mehan and Oohan. 1973: Sanchis el al., 1984). These differencesprobably reflect differences among phylogenetically diverged groups which may be identifiedless ambiguously by sclerotial morphology (Cotty. 1989a). However. in strains that dopnxtuce both sclerotia and aflatoxins. there appears to be an interrelationship betweenregulation of aflatoxin biosynthesis and regulation of sclerotial morphogenesis (Colty. 1988).This is suggested by: A) association of increases in aflaloxin production with inhibition ofsclerotial maturation when cultures are exposed to cither acidic pH or fungicides which inhibitergosterol biosynthesis (Cotty. 1988: Bayman and Cotty, 1990); B) coincidence of sclerotialmaturation with cessation of aflatoxin production (Cotty. 1988); C) high aflatoxin content ofsclerotia (Wicldow and Cole; 1982, Wicklow and Shotwell. 1982: Cotty, 1988): D) possibletranspon of aflatoxins from mycelia into sclerotia (Cony, 1988; Bayman and Cony, 1990),These observations suggest that sclerotium maturation is associated with a signal thatterminates aflatoxin biosynthesis. Delays in sclerotial maturetlion may thus delay thetennination signal and be associated with increased aflatoxin concentrations.

The interrelationship between sclerotial morphogenesis and aflatoxin biosynthe.~is issupported by recent advances in our understanding of the molecular biology of aflatoxinproduction. During characterisation of genes involved in aflatoxin biosynthesis. influencesof specific genes on both biosynthesis and morphogenesis has been observed (Skory et aI.,1992). However. as Skory et al. (1992) point out. it is not clear whether this relationship isa din:ct influence of either aflatoxins or aflatoxin precursors on sclerotia or a regulatoryassociation. The recent isolation of a putative regulatory element (apa-2) that influences bothprocesses (Chang el al., 1993) also corroborates the relationship. The suggestion of Skorytt 01. (1992), that aflatoxins themselves may serve a regulatory role during sclerotia!development. is interesting in light of the ability of aflatoxin B l to directly bind DNA(Muench et al.• 1983); a regulatory role for aflatoxjns in mature sclerotia is also possible.

,.

Accumulalion or Analo"ins in Subslrales. Aflatoxins are a concem in agriculturebecause large quantities can accumulate in certain plant materials. This accumulation maybe a survival adaptation directed at either preventing ingestion of infested seed or inhibitingcompetition (Janzen, 1977; Bilgrami and Sinha. 1992). However. large quantities ofaflatoxins are not accumulated in many plant part... in which the fungus increases and ismaintained (Griffin and Garren, 1976: Takahashi et 01.• 1986). and A. flavus is not veryefficient at eilher degrading aflatOxins or convening them to use (Doyle and Marth. 1978).Many strains of A. flavus do not produce large quantities of aflatoxins (Davis and Oeiner.1983) and when grown with other microbes (which is common in nature) toxin productionis greatly curtailed (see Strain Isolation and Accumulation of Aflatoxins). Indeed. it mightbe argued that most materials in which A. flavus grows and is maintained. are notcontaminated with large quantities of aflatoxins. If accumulation of aflatoxins in plantsubstrdtes is a directed fungal strategy. it is a very inefficient one. Accumulation may beinadvertent. caused by interference with sclerotial morphogenesis (Cotty. 1988). Export ofaflatox:ins from 'producing cells (Shih and Marth. 1973) might be directed at creatingaccumulations in the sclerotial rind (Willens and Bullock. 1992). Accumulation of aflatox:insinlracellularly and in dead cells of the sclerotial rind is testable by histological techniques.

Microbiallnleractions and Analoxin Biosynlhesls. Aflatoxin biosynthesis is readilyinhibited by microbial competition. Many microbes interfere with aflatoxin production inculture (Kimura and HirdOo. 1988: Roy and Chourasia, 1990) and in crops (Ashworth et of.•1965; Ehrlich et at.• 1985). Even A. flavus and A. parasiticus strains and/or mutants whichdo not produce aflatox:ins can interfere with aflatoxin production and/or crop contamination(Ehrlich. 1987; Cotty. 1990; Brown et oJ., 1991). Interference apparently occurs throughcompetitive ex:clusion (Cotty et 01.• 199(0). production of interfering compounds (Shantha et01.• 1990) and/or competition for nutrients (Cotty et al.• 199(0). Isolation of aflatoxin­producing strains from interfering strains is thus prerequisite for accumulation of highaflatoxin concentrations in crops (Bullerman et 01 .• 1975; Roy and Chourasia. 1990).Isolation may occur spatially. as occuno in labordtory tests on sterilised substrates. orphysiologically. usually when temperature or substmte moisture or composition favourdominance of A. flavus. Strain isolation may be one mechanism through which bothwounding during crop development and high temperdture favour very high toxin levels; rapidwound colonisation with aggressive invasion of developing tissues may pennit such isolation.If isolation of toxigenic strains is inadequate. poor aflatoxin production will result eventhough conditions favour growth and reproduction of the inciting fungus (BuIJennan et at.•lQ'7l:;)

Inleractions with Hosts. Aflatoxins are toxic and exert several physiologic effectson most hosts of the A. flavus group including plants. insects and mammals (Roberts andYendoI. 1971; Mclean et 01.. 1992; Robens and Richard. 1992). Aflatoxins may thus mediatepathogenesis either as a determinant of strain pathogenicity or by increasing so-ain virulence.Several lines of evidence suggest that this is. at least. not always the case. Even though veryhigh toxin levels have been detected in plants (over 500 mgkg· l

) (Lee et at.• 1990» andinsects (over 13 mgkg'/) (Ohtomo et al.• 1975). A. flal'us group isolates from insects.mammals. and plants may either produce aflatoKins or not. Furthermore. isolates which donot produce toxins retain ability to cause disease in the evaluated hosts (Cotty. 19890;Drummond and Pinnock. 19(0). In insects. aflatoxins may serve as virulence factO~.

increasing the rate at which infected insects die (Ohtomo et al.. 1975). However, the abilityof A. flavus to infect and invade insects appears to be more dependent on enzymaticdegradation of host proteins and cuticles (Sussman. 1952; Charnley. 1989).

,.

Conservation or Anatoxin producing ability. The A.. flovus group is a mosaic ofnumerous strains delimited by a vegetative incompatibility system (Bayman and Cotty. 1991).These strains appear to evolve. at least in general. as clones (groups of identical organismsdescendr.d from a single conunon ancestor by milosis) (King and Stansfield. 1985). Theseclones may move rapidly from rare 10 frequent depending on opponunity (Bayman and Cotty.1991). The importance or a given trait 10 strain success may be viewed both by the diversityof strains expressing that uait and the frequency of the expressing Slrains. Consistentexpression of a uail by diverse strains may indicate that trait is beneficial in multiple nichesor that it has use in a broad ecological niche. The tendency to produce aflatoxins is highlyvariable within the overall A. flavus group. However. loxin pnxluction is more consistentamong strains which are more closely related on the basis or morphological. physiological.genelic, or molecular characteristics (Domer et 01.• 1984; SailO et 01.. 1986; Cotty. 19890:Moody and Tyler. 199Oa.b: Egel and COllY. 1992; Bayman and Cotty. 1993). Thus. we canl$sociate conservation of loxin production over eVOlutionary time with cenain clusters ofstrains and loss of loxin pnxlucing ability with others. These observations may lead to newinsights on potential adaplive values of aflatoxins as we learn more aboul the basic biologyof the various clusters. Aflatoxigenicity is highly conserved among most wildtype strains ofA.parasiticus and A.fIovus suain S (Domer et of.• 1984; Cotty. 19890). Anatoxin-producingability is readily losl in culture and thus, conservation among fltld isolates of theseevolutionarily diverged cJusaers implies a slrOng selective force causing retention ofafla.lOxin·producing ability. These clusters may share a common use for aflatoxins or may each havedifferent uses. S strain isolales are more closely related 10 L suain isolates than to A..porasiticus (Egel and Cotty. 1992: Bayman and Cony. 1993). Apparently divergence of theLandS strains is relatively recent compared to divergence of A. flavus and A. parasiticus.Unstable toxin production (Boller and Schroeder, 1974; ClevSD'Om and Ljunggren. 1985) andreduced toxin producing ability are characteristics of the L suain (Cotty. 1989a). and Baymanand Cotty (1993) found that low toxin producing suains within the L suain are more closelyrelated to atoxigenic sttains than to highly IOxigenic strains. Thus. aloxigenicity apparentlycan be a multistep process in this group and for at leaS! one of the ecological niches 10 whichthe L so-ain is adapted, aflatoxins do nOl confer an imponant advantage. Aflatoxins do notincrease fungal virulence 10 crops (Cotty. 19890) and atoxigenic A.flavus strains have beenassociated with aerial crop parts (Bilgrami and Sinha. 1992); A. parasiricus (Davis and [)einer1983) and S strain isolates have been associated with a soil habital (see Diversity ofEcological Niches). Thus. the soil environment may favour conservation of toxin productionand the aerial environment may nol.

In addition to high aflatoxin pnxloction. reduced virulence to plantS is also associatedwith the S strain of A.flavus (Cony, 19890; Cony et 01.. 199Ob). Reduced viruJence sternsfrom failure to produce the most active A.flavus pectinase (Cony et at.• I99Ob: Cleveland andCotty. 1991; Brown et 01., 1992). Low virulence. stenvning from reduced ability to decayand colonise plant tissues (Brown et af.• 1991) may imply adaptation 10 a niche where suchtraits are nOt essential. Thus, high anatoxin producing ability is associated with strainsadapted to ecological niches where infection of crops is probably not essential.

Accumulation of large quantilies of G anatoxins by A. nomius, A. parasiticus, andeenain S strain isolates (Hesseltine et at., 1970; Domer et 01.• 1984; Saito et 01., 1986;Kurtzman et al., 19&7), but nOI by other A. flavus isolates. provides an additional puuJe.Unique activities have nOI been associated with G aflatoxins. 1berefore, it's difficult toenvisage selective advanlages conferred by relenlion of G aflatoxin producing abilily.Production of G aflatoxins may merely renect slight differences in pathway regulation(Bhatnagar et 01.. 1992) or retention of an ancestral uait

17

SELECTION OF ASPERGILLI ASSOCIATED WITH AGRICULTURE

Strain Sdtdion in the Past

A.. flavllS group populations have generally been altered by agriculture throughdisruption of habitat and introduction of nutrients. Production of crops under environmentalconditions conferring a competitive advantage 10 these fungi pennits lheir rapid incn:ase oncrop resources (Griffin and Garml. 1976; lee ~t oJ.. 1986). After cropping. large quantitiesof crop remnants and debris are incorporated into field soil; additional remnants remainingafter crop processing (from sorting. ginning, paring. shelling. ctc.) are also often incorporated.This organic mauer may both be superficially associated with large quantities of A.. fIavusgroup propagules and heavily colonised (Stephenson and Russell. 1974; Griffin and Garren.1976). Under conditions panicularly favourable to A.flavus. most organic debris incorporatedinto the soil can be colonised by A.flavus (Ashwonh et al.• 1969). Strains associated withthe crop and debris 3fe diverse and generally not deliberately selected (Bayman and Cotty,1991). Deliberate selection of specific strains with particular- characters has been forproduction of enzymes for me European baking industry (Barbesgaard er 01.• 1992) and forproduction of traditional fennentation products in the orient (Beuchat, 1978).

Fungal selection has reduced strain toxicity (Kunzman eloJ.. 1986) and increasedfungal traitS associated with both product quality and efficient fennentution (Wicklow, 1982.1990). Use of these fungi over centuries has inadvenently resulted in the release of largequantities of !;pores and colonised organic debris (Wicklow. 1990: Barbesgaard et aJ, 1992).Such strain selection and release may have altered A. flavus populations in the vicinity ofindustries and may partly explain strain distribution (Manabe et ai" 1976). The validity ofthis speculation might be teSted with recently developed techniques to characterise andcompare strUctures of A.flo\'us populations (Bayman and Cotty. 1991. 1993).

The Potential of Strain Selection

There are no methods for preventing aflatoxin contamination that are both reliable andeconomical. To fully protect crops from contamination. procedures muSt be active in the fieldunder hot. dry conditions that are not very conducive to crop development but, often are nearoptimal for A. flavus group fungi. Controls must be effective during both the cropdevelopment and post-maturation phases of contamination cycles. The procedure must fitwithin agriculture's economic constr.lints and for worldwKle use. must be effective undersuboptimal storage conditions and with low technological input. Furthermore. because mostcontamination occurs in damaged seed (which for many crops eimer cannot be soned out ormust be used) controls must prevent contamination of plant pans compromised by eitherphysiological StresS or predation. These are difficult requirements for II. procedure directedat preventing the relatively Idre. highly contaminated seed.

A promising avenue of control. that may meet the above criteria. is the seeding ofagricultural fields with atoxigenic A. jlOVIIS group strains in order to reduce toxigenicities ofresident populations (Cotty 1991b.I992a). A.flovus does not require aflatOldns to infect cropsand production of large quantities of aflatoxins in crop partS does not increase either strainvirulence or strain ability to colonise and utilise crop resources (Cotty, 19890). This led tospeculation that applied atoxigenic strains might outcompete toxigenic strains during cropinfection and thereby reduce conramination (Cony. 19890; Cole and Cotty, 1990).Greenhouse and field experiments in which either developing conon bolls or developing comears were wound inoculated with various strain combinations demonstrate the potential ofatoxigenic strains to reduce contamination (80 to 90%) during crop development (Cony, 1990;Brown tloJ., 1991). Individual crop components are often coinfected by multiple A.flo\lusstrains and cottonbolls damaged by pink bollwonns are infected by A. flavus strains in

18

multiple vegetative compatibility groups at least 50 to 80% of the time (Bayman and Cotty,1991; PJ. Cony. unpublished). Pink bollworm-damaged bolls contain the majority ofaflatoxins in commercial fields (Cotty and Lee. 1989). Thus. the abilily of atox..igenic strainsto interfere with contamination in co·infected bolls may be ofreal practical value. Atoxigenicslllins n:duce contamination by both spatially excluding toxigenic strains and by competingfor resources required for production of aflatoxins (Cony t!t al. 199(0). However, not allatOxigenic sb'ains are capable of reducing contamination during co-infection (Cotty, 19914):thus SlJ1l,in optimisation in co-inoculation teSts should be pre-requisite to field evaluation.

In theory. seeding fields with atoxigenic strains relatively early in crop developmentmay pennit seeded strains to compete with other resident strains for crop associated resources(Cole and Cotty. 1990). The seeded SbllinS may thus increase in population size along withtoxigenic strains when environmental conditions favour aflatoxin contamination (Cole andCotty, 1990; Cotty, 1992a.c). At the same time, lhe atoxigenic strains may compete forinfection sites. In special environmental contrOl plots, Domer et al.. (1992) havedemonstrated that A. parasiticus strains which accumulate specific aflatoxin precursors (Le.a native strain that accumulates Q-methylslerigmatocystin and a mutllnt that accumulatesversicolorin-A) but nOt· aflatoxins. can interfere with aflatoxin contamination whenconcentrated propagule suspensions are applied to developing peanuts. Applications resultedin long term (several years) fungal population changcs and support the use of atoxigenic A.{/tNWS group strains in preventing contamination of peanuts. The most comprehensive fieldtests. to date, have been performed on COllon grown in Yuma County. Arizona, This area hasthe moSI consistent aflatoxin contamination of cottonseed in the United States (Gardner et al.,1974). An atoxigenic strain was seeded on colonised wheat seed (Cony, 19920) into a fieldof developing COlton, prior to crop flowering (COlty. 1991b). The distribution of vegetativecompatibility groups (VCGs) within this field had been detennined in previous years,(Bayman and Cotty. 1990) and a strain in a rare VCG was seo:1ed. Five months later thecrop was harvested and the distribution of lhe applied strain on the crop was detennined bymutating isolates 10 nitrate auxotrOphy and assessing VCG. Strain seeding resulted in largeand signiflCaJ1t reductions in the aflatoxin Content of the crop at maturity and aflatoxin contentwas inversely correlated with the incidence of the seeded VCG (Cony. 1991b). Similar tests.performed in subsequent years, also demonstrate that alOxigenic strains applied early in cropdevelopment can panially competitively exclude toxigenic strains and thereby reducecontamination (Cony. 1992c); [his early suain application is associated with neither increasedcrop infection nor increased A.flavus populations on the crop at maturity.

The theoretical advantage of atoxigenic strains of A.jlavus over other microorganismsthat mighl be used to competitively exclude aflatoxin-producing stra.ins is that atoxigenicstrains are apparently adapted to similar environmental conditions as toxigenic strains. Otherpotential agents. such as bacteria (Kimura and Hirano, 1988; Bowen et al. 1992), may beinactive under the hoI, dry conditions associated with aflatoxin contamination. The use ofalOxigenic stn!ins seeks to limit neither the amount of crop infection by the A. flavus groupnor the quantity of these fungi associated with the crop. The procedure merely selects whichfungi become associated with the crop. Thus, crop quality losses typically associated withfungal infection (i.e. increased free fatty acids) will not be ameliorated. Seeding aloxigenicsaains might not result in increased crop infection because infection is more heavilydependent on host predisposition and the environment than on the number of propagule.'i ofA. flavus. Indeed in three years of tests on cotton, seeding has nOt resulted in increasedinfection rates (Cotty, 1992c). However, under certain citcumSlances with sufficiently lowinitial A.flavus levels and sufficiently high seeding rales. increased infection rates in tmltedcrops might be expected. However. A. flavus typically decays predisposed crop componentsthat. under different environmental conditions, would be infected by other microbes. Thus,these infections probably would nOI be: of a magnitude to cause concern.

Populations of A./lavus increase on crops very rapidly under conditions favouraMe

,.

to contamination. The ultimate magnitude of the A. j10vus group is largely dependent on theav!ilabk resources and the environment ThUs. even in areas with perennially low aflatoxinconlamination. high A.flovus populations can rapidly develop during droughts (Shearer ~t a/.,1992): lIle composition of these rapidly increasing populations might be partially controlledby properly timed seeding.

A.flovus group fungi typically become associated with crops in the field during cropdevelopment and remain associated with the crop during harvest, storage and processing.Thus, seeding of atoxigenic strains into agricultural fields prior to crop development mayprovide postharvest protection from contamination by associating the harve.'ited crop with highfrequencies of atoxigenic Smllns. Atoxigenic strolins applied both prior to harvest and afterharvest have been shown to provide protection from aflatoxin contamination of com (Brownel al.• 1990, even when toxigenic strains are associated with the crop prior to application.

Domestication of A.flovus group fungi for seeding into agricultural fltlds may causesome concern over the pathogenic potential of these fungi to humans (Pore et al.• 1970).Although limiting exposure of high risk individuals to aspergilli will reduce infection risk.particularly in hospitals. it might be argued that host predisposal is more imponant indetennining disease incidence than exposure to fungal propagules (Wardlaw and Geddes,1992: St Georgiev. 1992; Rinaldi, 1983). In many agricultural industries and communities.workers and residents respire high conCentrdlions of Aspergillus spores. Clearly suchexposure is undesirable, but such respiration may occur without noticeable disease. This pointis particularly clear for fungal strains used to produce koji and baking or brewing enzymes(Barbesgaard et al., 1992). In these industries. generations of workers have been exposed10 very high concentrations of spores tliroughout their working years witli a very lowincidence of disease (Barbesgaard t!t al.• 1992). Barbesgaani et al. (1992) argues for A.oryzat to be classified as "Generally Regarded As SafeM (GRAS). partially on this basis.

Seeding agricultural fields with select fungal isolates can result in A.fla\lus populationswith altered composition. but without increased population size (Cotty, 1991b, Cotty 1992c).Thus, seeding may provide the opponunity to improve the overall safety of fungal populationsby reducing human exposure to anatoxins through both dietary and respiratory routes (seeEffects of Anatoxins on Humans and Domestic Animals). The frequency of fungal traitsother than anatoxin-producing ability mighl also be altered and. in so doing, fungal virulenceto animals might be reduced or fungal sensitivity to therapeutic agents might be increased(Cotty and Egel, 1992). Other fungal traits delrimenwlo humans or human activities (i.t!.allergenicity) might also be minimised and beneficial trailS (~.g. ability to decay crop debrisbetween plantings) might be maximised. The concept of fungal seeding also applies to fungiother than A. fl(Nus, particularly to other aspergilli. A. fumigotus. a more potent animalpathogen than A. fl(Nus. is a very frequent degrader of plant debris (Gando))a and Aragno.1992). Extremely high concentrations of A. /umigorus spores may be associated withcomposting organic maner (Gandolla and Aragno, 1992). It may be possible to select strainsof A. fumigolus. in II manner similar to A. /1011/4:,., in order 10 optimise both safety anddecomposition.

This strategy of seeding fields with select strains of A. navus has drawn repeatedcontroversy and criticism ba.'iC:d on the dangers of A.j1avus populations (Wicklow. 1993;Kilman, 1993). However. the choice presented is not whether or not there will be fungi. Thechoice is whether we will detennine. through deliberate selection. which strains make up thepopulations. C'wTent agricultural practice does seed fields with very large quantities oforganic matter colonised with A. flavus group fungi. This material is in the fonn of cropremnants. gin tra.~h. com cobs, etc. It is common practice to incorporate such materials intofield soils. This differs from the seeding strategy suggested here in that seeded strains arenOI selected, the quantity of material incorporated is very large, and incorporation is not timedto give applied strains preferential exposure to the developing crop.

20

Ecological Significance

The use of atoxigenic strains of A. flavus to control aflatoxin production has beenhindered by a lack of infonnation about fungal population biology. In many ways this fieldlags rwenty or thiny years behind comparable studies on animals and plants (Burnett. 1983);lhe best-known fungus in this respect is probably Nturorpora crassa (Peritins and Turner,1988). Dispersal. change in population structure over time. and natunll selection are poorlyunderstood in fungal populations. This panly results from difficulty in tracking individuals(McDonald and Martinez.. 1991). Interactions between conspecific genetic individuals havenot been widely regarded until recently (Rayner. 1991). Funhennore, studies on one groupof fungi have often tumed out to have limited application to other groups. AU these problemsue complicated in fungi like the A. j1tJ\1US group by tremendous reproductive and dispersalabilities, the lack of a known sexual stage. parasexuality (Papa, 1984), and mitoticchromosomal rearrangements (Keller tf al., 1992).

During the coune of experiments discussed here, data has been collected on variationin many characters in many natural isolates. Areas have been sampled repeatedly over severalyears and known isolates have been introduced into fields and their survival and dispersalfollowed over the course of years; this has not been done with Neurospora. This body ofdata on how A.flavus genetic individuals survive. spread, and inleract, may tum oul to be asinteresting as the biocontrol strategy it was designed to support.

One concepl in sustainable agriculture is to "srudy the forest in order 10 fann like theforest~ (Jackson and Piper, 1989). Understanding distribution, variation, and competition infungal populations in nature and agriculture may lead to successful use of this principle.

ACKNOWLEDGMENTS

We are grateful to Mrs. Darlene Downey, technical assistant to P.J. Cotty, for hercontributions 10 the work discussed here.

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