APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1992, p. 809-8140099-2240/92/030809-06$02.00/0Copyright X) 1992, American Society for Microbiology

Genetic Analysis of the Role of Phytoalexin Detoxification inVirulence of the Fungus Nectria haematococca

on Chickpea (Cicer arietinum)V. P. W. MIAOt AND H. D. VANET ENt*

Department ofPlant Pathology, Cornell University, Ithaca, New York 14853-5908

Received 28 May 1991/Accepted 5 November 1991

Chickpea (Cicer arietium L.) produces the antimicrobial compounds (phytoalexins) medicarpin andmaackiain in response to infection by microorganisms. Nectria haematococca mating population (MP) VI, a

fungus pathogenic on chickpea, can metabolize maackiain and medicarpin to less toxic products. Thesereactions are thought to be detoxification mechanisms in N. haematococca MP VI and required for pathogenesisby this fungus on chickpea. In the present study, these hypotheses were tested by examining the phenotypes ofprogeny from crosses of the fungus that segregated for genes (Mak genes) controlling phytoalexin metabolism.Maki and Mak2, two genes that individually confer the ability to convert maackiain to its la-hydroxydienonederivative, were linked to higher tolerance of the phytoalexins and high virulence on chickpea. These resultsindicate that this metabolic reaction is a mechanism for increased phytoalexin tolerance in the fungus, whichthereby allows a higher virulence on chickpea. Mak3, a gene conferring the ability to convert maackiain to its6a-hydroxypterocarpan derivative, also increased tolerance to maackiain in strains which carried it; however,the contribution ofMak3 to the overall level of pathogenesis could not be evaluated because most progeny fromthe cross segregating for this gene were low in virulence. Thus, metabolic detoxification of phytoalexinsappeared to be necessary, as demonstrated in the Makl and Mak2 crosses, but not sufficient by itself, as in theMak3 cross, for high virulence of N. haematococca MP VI on chickpea.

Phytoalexins are low-molecular-weight antimicrobial com-pounds which are produced and accumulated by plants whenthey are attacked by microorganisms (2, 15). One type ofexperimental support for the importance of phytoalexins as a

component of disease resistance emerged from studies of therelationship between pathogenicity and phytoalexin detoxi-fication in fungi (18). For example, naturally occurringisolates of the ascomycete Nectria haematococca matingpopulation (MP) VI Berk. et Br. (asexual state; Fusariumsolani) vary in their ability to attack pea (Pisum sativum L.).Isolates that cannot detoxify pisatin (Pda-), the phytoalexinproduced by pea, are more sensitive to pisatin in vitro, andthe most virulent isolates on pea are Pda+ (19). Similarly,natural variants of the potato pathogen Gibberella pulicaristhat do not metabolize the potato phytoalexin rishitin aremore sensitive to this compound and cause less tuberdamage than those that do metabolize it (5). Subsequentgenetic studies showed that such detoxification is tightlylinked to expression of high virulence in each of thesehost-pathogen interactions (18).A study by Lucy et al. (11) of a root rot disease of

chickpea (Cicer arietinum L.) that is also caused by N.haematococca MP VI suggested that metabolism of thechickpea phytoalexins (-)-maackiain and (-)-medicarpin (8,9) could again be important for pathogenesis; virulence on

chickpea among 130 field isolates examined in that study wascorrelated with their ability to metabolize maackiain.Three genes for metabolism of maackiain in N. haemato-

cocca MP VI are known (14a). The Makl and Mak2 genes

* Corresponding author.t Present address: Institute of Molecular Biology, University of

Oregon, Eugene, OR 97403-1229.t Present address: Department of Plant Pathology, University of

Arizona, Tucson, AZ 85721.

are individually able to direct conversion of maackiain to itsla-hydroxydienone (laHM) derivative. The Mak3 gene con-trols conversion of maackiain to its 6a-hydroxypterocarpan(6aHM) derivative. An isolate of the fungus performed thesame reaction on both chickpea phytoalexins (4), so it islikely that these genes are involved in modification of bothmaackiain and medicarpin.The reactions controlled by the Mak genes constitute the

first step in two of the three pathways of maackiain andmedicarpin metabolism in N. haematococca MP VI (14a).These modifications are presumed or have been shown to becatalyzed by monooxygenases (4, 14, 18) and are associatedwith detoxification. For example, the la-hydroxydienoneand isoflavanone derivatives of medicarpin were less toxic toN. haematococca MP VI than the parent compound (3),while the 6a-hydroxylation of medicarpin and maackiain bySclerotinia tnfoliorum and Botrytis cinerea (12) and of therelated phytoalexin phaseollin by Septoria nodorum (1) andother fungi (6) also resulted in detoxification. The la-hydrox-ydienone derivatives of both phytoalexins have been foundin chickpea stems infected with an isolate of N. haemato-cocca MP VI that performs this reaction (3), indicating thatphytoalexin metabolism by the pathogen occurs in situ.The identification of Mak genes allows further evaluation

of whether the metabolism of maackiain is a detoxificationprocess and whether it is required for high virulence of N.haematococca MP VI on chickpea; if there is a causalrelationship between phytoalexin-metabolizing ability, toler-ance, and virulence, then these traits would appear to begenetically linked, i.e., would be expected to cosegregate.The goal of this study was therefore to examine the role ofphytoalexin detoxification in the chickpea-N. haematococcaMP VI interaction by determining the association of maack-iain metabolism, maackiain tolerance, and virulence instrains derived from crosses segregating for Mak genes.

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MATERIALS AND METHODS

Fungi. Field isolates T-126, T-161, T-200, and progeny

derived from them were described in a previous investiga-tion that identified Makl, Mak2 and Mak3 (14a). T-161 andT-200 metabolize maackiain and medicarpin (Mak+), whileT-126 does not (Mak-). T-161, the source of Makl andMak2, converts maackiain to laHM, whereas T-200, thesource of Mak3, converts maackiain to 6aHM. The Mak+phenotype of T-161 is also referred to more specifically as

1aHM+, and that of T-200 is more specifically 6aHM+. Thepedigree of progeny derived from these isolates is indicatedin the strain identification number; e.g., strain 156-30-6specifies cross number 156 and ascus number 30, and theascospore number, 6, is arbitrary, because tetrads of N.haematococca MP VI are unordered.

Additional field isolates of N. haematococca MP VI usedin this study were obtained by screening Fusarium solaniisolates (collected by A. T. Casas, Finca "Alameda delObispa," Cordoba, Spain) from diseased chickpea and iden-tifying those which mated with reference strains of N.haematococca MP VI by protocols described previously(17). All fungi were maintained on V8 Juice agar (17) andstored as slant cultures at 4°C or as conidia in 50% glycerolat - 138°C.

Plants. Seeds of ICC12246 (ICC2883), a desai chickpeabreeding line, were obtained from M. P. Haware at theInternational Crop Research Institute for the Semi-AridTropics, Hyderabad, India.

Chemicals. Maackiain and medicarpin were extractedfrom red clover roots and from fenugreek seedlings, respec-

tively (3, 11). 6a-Hydroxymaackiain and la-hydroxymaack-iain were obtained by metabolism of maackiain by N.haematococca MP VI (4). Pisatin was extracted from pea

seedlings (16), and [3-0-methyl-14C]pisatin was prepared byfungal demethylation of pisatin, followed by chemical meth-ylation with "4CH31 (19). Phytoalexins were added to cul-tures from 50x or 100x stock solutions in dimethyl sulfox-ide.

Phytoalexin metabolism assays. The ability of the newlyidentified field isolates of N. haematococca MP VI tometabolize maackiain was scored by growing the fungi in thepresence of phytoalexins and then assaying for maackiainmetabolites in culture fluids by thin-layer chromatography(TLC) (4, 14a). Their ability to remove the radiolabeledmethyl group of [3-0-methyl-14C]pisatin while growing on

agar medium was assayed as described elsewhere (13).Progeny of crosses were scored previously for these traits(14a).

Phytoalexin tolerance assay. Sensitivity to maackiain andmedicarpin was assessed by mycelial growth on phytoalexin-amended PGA, an agar medium containing peptone andglucose (11). The difference in colony radius after 5 days ofgrowth on medium amended with maackiain (50 ,ug/ml) and2% dimethyl sulfoxide versus solvent control medium (2%dimethyl sulfoxide) was expressed as percent inhibition. Theaccuracy of a single measurement of percent inhibition wasabout +5%, e.g., five Mak+ isolates assayed once on each offive or more occasions showed mean inhibitions of 6.3 to10.4%, with standard deviations (SD) of 2.2 to 3.8%, whilefour Mak- isolates assayed once on six or more occasionsshowed mean inhibitions of 23.1 to 30.5% and SD of 3.1 to4.4% inhibition. Variation within an experiment was essen-

tially the same as that shown above between experiments,e.g., for two of the Mak+ isolates when they were testedwith five or six replicates in a single experiment, while a

third showed a small difference (mean = 6.3%, SD = 3.3%between experiments versus mean = 7.6%, SD = 1.7%inhibition within an experiment).

Field isolates, parent strains, and random ascospore prog-eny were tested at least twice for tolerance to maackiain;progeny collected as tetrads were tested only once becausetetrads contain pairs of twins (see Results). Progeny fromcross 156 were tested against medicarpin at 120 jig/ml, but as70 ,ug/ml also gave a consistent differential response, thelower concentration was routinely used for other isolates.Only field isolates were assayed more than once for toler-ance to medicarpin.Virulence assay. Virulence was determined in a manner

similar to that described previously (11) with some modifi-cations. Chickpea seeds were washed in running tapwaterfor 1 to 3 h before being planted individually in test tubescontaining sterile vermiculite and Hoagland's solution (7). Aplastic collar was placed around the mouth of each test tube,creating a 2-cm extension into which additional vermiculitecould be added to cover each seed. Tubes were placed inglass tanks containing 2 to 3 cm of water to maintainhumidity, and tanks were covered with plastic wrap andblack cloth. Plants were grown at 25 to 27°C for 5 days in thedark and then exposed to continuous light for the rest of theexperiment. At 1 week, the plastic collar was moved aside(excess vermiculite was removed), and a small puncturewound was made with a dissecting needle in the stem 1 cmabove the cotyledons. A 3-mm disk of mycelium from a2-day culture on PGA was placed over the wound, and thecollar was moved back into position.Each isolate was inoculated on eight plants. One set of

seedlings inoculated with a known virulent isolate wasincluded in each tank as a positive control. Plants were heldat 20 to 22°C in the tanks for 4 to 5 more days, and then thelength of the lesion at each inoculation site was measured. Inthis assay, lesions observed were more uniform when inoc-ulated with some strains than with others. While somestrains regularly showed little ability to form lesions (meanlesion size of <2 mm), and others, such as T-200, consis-tently formed large lesions (mean lesion length of T-200 was11.9 ± 2.2 mm over eight experiments), there were alsostrains that produced low to intermediate levels of diseaseoverall but still showed perhaps one or two intermediate tolarge lesions among the eight replicates. It should be notedthat seeds from chickpea breeding lines, while not geneti-cally uniform, are routinely bulked at harvest; consequently,seedlings in the virulence assay may harbor differences insusceptibility to disease which are not obvious if the inocu-lated strain has very high or very low pathogenic potential,but which become noticeable if the strain used has interme-diate pathogenic abilities. This is a possible reason for highvariation among the eight replicates inoculated with any oneisolate. However, the genetic heterogeneity of the plantmaterial is presumably randomly distributed as far as theseexperiments are concerned, so the overall effect of the fungalgenes being tested should still be observable when the dataset is sufficiently large. That the plant material is heteroge-neous but not known to be biased also allows some statisticaltests to be applied (see Results).Only some progeny from each cross were tested for

virulence because this line of chickpea seeds was available inlimited quantity. In cross 156, 18 random progeny weretested. In other crosses, only enough of the progeny fromeach tetrad were tested to be certain of including all fourmeiotic products; these were identified from the segregationof other markers (14a).

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PHYTOALEXIN DETOXIFICATION AND VIRULENCE 811

TABLE 1. Characteristics of N. haematococca MP VI isolatesfrom diseased chickpea

% Inhibition ofMak Growtha (mm) growth by: Mean lesion

Isolate pheno- on control length (mm)type medium Maackiain Medicarpin + SD

(50 ,ug/ml) (70 ig/ml)T384 + 20.7 6 7 5.8 + 2.64T385 + 20.6 8 2 4.2 ± 0.90T387 + 20.4 7 9 3.1 ± 2.90T389 + 19.3 2 5 9.6 ± 3.20T390 + 22.3 -1 0 2.7 ± 4.52T392 + 23.2 13 4 5.2 ± 5.91T383 - 20.5 30 26 <2"T391 - 20.6 27 21 1.2 ± 0.51T386 - 19.1 30 22 NTT388 - 22.4 35 29 0.9 + 0.42

a Colony radius after 5 days; results of a second experiment were similar.b In a separate assay, isolate T383 formed only lesions of <2 mm on all eight

inoculated seedlings after 5 days; for comparison, isolate T389 was associatedwith a mean lesion length of 11.9 ± 4.10 mm in the same experiment.

NT, not tested.

RESULTS

Phytoalexin metabolism, sensitivity, and virulence in fieldisolates. Only 3 of 130 isolates of N. haematococca MP VIexamined in the previous survey were isolated from chick-pea (11); therefore, additional isolates from this host weresought. Among 10 new isolates of MP VI identified bymating tests from this source, there was a strong associationbetween an isolate's ability to metabolize maackiain and itstolerance of phytoalexins (Table 1). All Mak+ isolates wereless inhibited than all Mak- isolates on phytoalexin-amended media, even though they grew equally well oncontrol media. The maackiain metabolite produced by Mak+isolates appeared to belaHM, based on relative mobility inTLC. Although the lesions caused by a particular isolatevaried in appearance and size, Mak- isolates most oftenproduced inconspicuous light brown lesions (<2 mm) at thesite of inoculation (a reaction type frequently obtained withwounded but uninoculated seedlings), while Mak+ isolatesproduced lesions that ranged from the <2-mm type to somethat were blackish brown (indicating greater necrosis of thehost) and girdled the seedling. Large lesions, when theyoccurred, were invariably associated with Mak+ isolates; inno instance was high virulence expressed by a Mak- isolate.In contrast to the previous finding that most (21 of 22) Mak-isolates of N. haematococca MP VI did not degrade the peaphytoalexin pisatin (11), two of the present four Mak-isolates (T383 and T391) were Pda-. The remaining twoMak- isolates and all Mak+ isolates were Pda+.

Phytoalexin metabolism, sensitivity, and virulence in iso-latesT-161, T-126, and their progeny. Most of the newlycharacterized isolates from chickpea were unable to functionas female parents, so they could not be crossed with eachother, and none crossed well enough with laboratory strainsto generate sufficient progeny for genetic tests. A successfulcross (cross 156) was found betweenT-161, a maackiain-tolerant, moderately virulent Mak+ isolate (original habitatunknown), andT-126, a moderately tolerant isolate fromsainfoin that was the most virulent Mak- field isolate previ-ously identified (11). Of 30 random progeny tested from thiscross, inhibition of radial growth ranged from 1 to 16%among the 24 Mak+ progeny and from 21 to 41% among the6 Mak- progeny (Fig. 1).

I)ia9

20

a |- Mak+|*T-161 0 Mak-

10 _4 40 T-;1426

0 0

.*e- * 0O^ * ^ A na.%A^ 110 10 20 30 40

% Inhibition by maackiain (50 gg/ml)so

FIG. 1. Virulence on chickpea, tolerance to maackiain, andmaackiain-metabolizing ability in isolates T-126, T-161, and theirprogeny (cross 156).

The preponderance of Mak+ progeny in cross 156 (14a)suggested that there was more than one Mak gene in T-161,and in fact this isolate was later shown to have at least twoMak genes, Makl and Mak2. The effects of these genes ontolerance did not appear to be additive, however, becauseT-161, at approximately 10% inhibition of radial growth (Fig.1), was not more tolerant than progeny strains which hadonly Maki or Mak2 (Table 2).

Eighteen progeny from cross 156 were tested for virulenceon plants. A similar range of virulence would be expectedamong Mak+ and Mak- strains if the Mak genes did notcontribute to virulence; however, high virulence appeared tobe associated only with maackiain tolerance and the Mak+phenotype (Fig. 1). We can apply a statistical evaluation ofthis conclusion by letting the mean lesion sizes of the Mak-progeny represent the threshold level of virulence achiev-able without Mak genes. Then, all other things being equal,progeny whose mean lesion size was at least 2 SD greaterthan that of the pooled Mak- average can be considered tobe more virulent than this threshold level. By this rationale,the eight Mak+ progeny with the largest mean lesion lengths

TABLE 2. Effect of Maki or Mak2 on growth ofN. haematococca MP VI

% Inhibition of radial growth byCross Parent Genotype maackiain (50 Kg/ml) in:aor pheno-

Mkno.a strains type Mak+ Mak Mak+ Makparent parent progenyb progenyb

230 156-30-6 MakI 6 5 ± 3 (67) 24 ± 4 (74)156-2-1 Mak- 31

263 260-1-1 Maki 8 6 ± 3 (42) 25 ± 4 (32)230-27-7 Mak- 28

272 156-30-6 Makl 9 3 ± 2 (35) 24 ± 2 (11)230-30-6 MakI 0

269 156-31-3 Mak2 15 12 ± 4 (27) 35 ± 8 (43)c230-31-1 Mak- 29

279 156-31-3 Mak2 9 10 ± 3 (30) 32 ± 3 (9)269-13-1 Mak2 12

a These crosses are described in the accompanying article (14a)."Mean ± SD calculated from the number of progeny shown in parentheses.'Includes both "normal" and unexpected Mak- progeny.

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812 MIAO AND VANEITEN

40

i0 __° °i. k

0~~~~~

0 20 40% Inhibition by medicarpin (120 gtg/ml)

FIG. 2. Tolerance of maackiain and medicarpin by isolatesT-126, T-161, and their progeny (cross 156). Correlation coefficient,R = 0.87. The group of progeny shown here overlaps but does notinclude all of those shown in Fig. 1.

(Fig. 1) are more virulent than their Mak- siblings (pooledmean lesion length = 2.43 mm, SD = 0.49 mm), presumablydue to the presence of Mak genes. The presence of thelow-virulence Mak+ strains argues that the Mak+ phenotypewas necessary but not sufficient for high virulence. Interest-ingly, many strains, including all of the Mak- progeny, were

less virulent than the Mak- parent T-126.Tolerance to maackiain versus tolerance to medicarpin.

Tolerance to maackiain was highly correlated with toleranceto medicarpin in the previous study (11), and this was againobserved among the new isolates from chickpea (Table 1),the progeny of cross 156 (Fig. 2), and strains used as parentsin other crosses (data not shown). Based on this correlation,progeny from later crosses were assayed only againstmaackiain.

Effect of Maki and Mak2 on tolerance to maackiain andvirulence on chickpea. Increased tolerance to maackiain wasassociated with both genes conferring the laHM+ pheno-type. This was determined by examining progeny fromcrosses between a Makl or Mak2 strain and a Mak- strain;Mak+ progeny derived from such crosses must carry anactive Mak allele, whereas Mak- progeny would not. Prog-eny carrying Makl from crosses 230 and 263 (both Makl xMak-) were approximately 20% more tolerant than theirMak- siblings (Table 2). Mak2 progeny of cross 269 (Mak2x Mak-) had a similar enhancement (Table 2).Unusual segregation of Mak genes in some crosses where

tetrad ascospores were collected provided an additionalopportunity to assess the role of these genes. A tetrad is aunit of genetic analysis dealing with segregation of traitsamong the four products of meiosis. In ascomycetes such asN. haematococca MP VI, the products of meiosis arerepresented by the set of eight ascospores (four pairs oftwins) in the ascus. If a cross involved two alleles at a singlelocus, e.g., alleles for Mak+ and Mak- at the Mak2 locus, a

progeny segregation ratio of 4 Mak+:4 Mak- in a tetradwould indicate that all copies of the Mak2 alleles have beentransmitted normally; aberrant inheritance can thus be de-tected if tetrads segregate in other than the expected pattern.While Mak2 segregation in cross 269 was generally normal,some tetrads had more Mak- progeny than predicted; thisbehavior did not appear to affect other loci, e.g., matingtype, which segregated normally in the same tetrads (14a).These unexpected Mak- strains were also more sensitive tomaackiain than their Mak+ siblings (Table 2).

Other examples of failure to transmit Mak genes normally

(A)

410.01-

oT ITetrad #

cross 230

: 0

0

8 8 0

0.1.I I

10 24

(B)

ii

.20

I-,

*a

.80

0 0

00 0

0

0

8

0

Ma-petp

cross 272

...Makl parent.I0 0

0

0

0

0 0

0 826 27 28 31 33 37 19 6 26

Tetrad#2 4 8 9 13 20 21

FIG. 3. Segregation for virulence on chickpea in tetrads from (A)crosses 230 (Makl x Mak-) and 272 (Makl x Maki) and (B) cross269 (Mak2 x Mak-). Each circle represents one member of thetetrad indicated on the bottom. Symbols: 0, Mak+ progeny; Mak-progeny. 0, Virulence of parent strains is indicated by dotted lines.

were observed in crosses 272 and 279; in these crosses, allprogeny were expected to be Mak+ because both parentscarried Makl or both carried Mak2 (14a). In every case,unexpected Mak- progeny were as sensitive to maackiain asMak- isolates from the field or from other crosses (Table 2).One representative of the aberrant Mak- progeny from

cross 272 was also tested for sensitivity to medicarpin (70,ug/ml). Growth of this strain was inhibited by 22% at thisconcentration of medicarpin, whereas inhibition of a Maklsibling and both Makl parents was only 8% + 4%. Thus, theloss of tolerance to maackiain appeared to be accompaniedby a loss of tolerance to medicarpin.

Virulence on chickpea cosegregated with the laHM+phenotype. The apparently higher virulence of Mak+ prog-eny than of their Mak- siblings (Fig. 3A) was supported bythe result of a paired-difference test with data from thetetrads in Fig. 3A and from three others not shown (a = 0.05;degrees of freedom = 10). In this test, each tetrad, repre-senting an independent meiotic event, was considered areplicate, and the difference in virulence between Mak+ andMak- progeny was tested for significant departure from zero(i.e., no difference in virulence associated with the presenceof Makl). In cross 272, unexpected loss of Makl wasaccompanied by a significant reduction in virulence (ao =

0.05; degrees of freedom = 2) in all cases examined (Fig.3A). Similarly, Mak2 progeny in cross 269 (Fig. 3B) weremore virulent than their Mak- siblings (paired-differencetest based on data shown for tetrads 2, 4, 8, 9, and 13 was

Mak2 paren

0 aa

50 85

-0 A --°---O Malckparent

0 00 80

e ' . . ..~~~0 9

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PHYTOALEXIN DETOXIFICATION AND VIRULENCE 813

significant at a = 0.05; degrees of freedom = 4); tetrads fromthe cross that contained only Mak- progeny also did nothave any highly virulent strains (Fig. 3B, tetrads 20 and 21).

Effect of Mak3 on tolerance to maackiain and virulence onchickpea. Evaluation of the relationship between Mak3,phytoalexin tolerance, and virulence in progeny of cross 241(between the Mak3 isolate T-200 and a Mak- strain) wascomplicated by (i) discovery of an additional uncharacter-ized means to metabolize maackiain, (ii) unexpected pres-ence of auxotrophy in some progeny, and (iii) general lack oftransmission of virulence to progeny.

Cross 241 produced not only progeny which were 6aHM+or Mak-, but also some which metabolized maackiain to anapparently novel product (14a). Among 22 tetrads assessedfor both Mak and sensitivity, Mak- progeny were the mostsensitive to maackiain (mean of 27% ± 1 SD of 4% for the 33progeny strains tested) and 6aHM+ progeny were the mosttolerant (11% + 3%, n = 78); interestingly, progeny thatproduced only the new compound also showed some toler-ance to maackiain (14% + 4%, n = 48). When a progenystrain from cross 241 that produced the new compound wasbackcrossed to its Mak- parent, increased tolerance againcosegregated with the ability to make the new compound: 11random progeny which made the new compound showedmore tolerance to maackiain (13% + 1%) than 13 Mak-siblings (23% + 3%).The contribution of Mak+ toward virulence could not be

measured among the progeny of cross 241 because moststrains had low virulence on chickpea. Of 44 prototrophicprogeny (representing 11 tetrads) tested, only 2 were mod-erately virulent (6.9- and 8.6-mm mean lesion length); bothof these were 6aHM+. The remainder of the progeny wereassociated with lesions of less than 5 mm even though theparent, isolate T-200, was consistently virulent (mean lesionlength over eight experiments = 11.9 + 2.2 mm). Assess-ment of virulence was further hindered by an unexpectedauxotrophy in many progeny (even though both parents ofthis cross were prototrophic) (14a). These progeny requiredan amino acid supplement for growth on minimal mediumand, upon inoculation on plants, caused only a local brown-ing reaction that appeared similar to the wounded butuninoculated control.

DISCUSSION

Hydroxylations at the la or 6a position and the ring-opening modifications of maackiain and medicarpin havebeen proposed as detoxification reactions in N. haemato-cocca MP VI because (i) la-hydroxylation and ring openingproduced derivatives of medicarpin that were less toxic toN. haematococca MP VI (3) and (ii) 6a-hydroxylation ofmedicarpin and maackiain by other fungi (12, 20) has alsoresulted in less toxic derivatives. The absolute linkagebetween the Mak+ phenotype and an increased tolerance tomaackiain in the present study is yet another indication thatthe la- and 6a-hydroxylations are detoxification mechanismsand contribute to the tolerance of N. haematococca MP VIto these phytoalexins.Denny and VanEtten (3) proposed that tolerance to the

chickpea phytoalexins in N. haematococca MP VI has twocomponents. A "nondegradative" mechanism appears toaccount for a basal level of tolerance in N. haematococcaMP VI that distinguishes it from other fungi, such as themorphologically similar N. haematococca MP I; e.g., themost sensitive Mak- isolates in MP VI are inhibited approx-imately 50% by 50 ,ug of maackiain per ml (11), whereas an

isolate of MP I is completely inhibited at the same concen-tration of maackiain (3). Tolerance above the basal level inMP VI is then afforded in some isolates by the degradativeability conferred by the Mak genes.The results of the present study suggest that degradative

tolerance is required, but insufficient per se, for high levelsof virulence. For example, while many Makl or Mak2strains were more virulent than Mak- strains, the existenceof other Mak+ isolates not pathogenic on chickpea indicatedthat traits in addition to the ability to detoxify chickpeaphytoalexins are needed for high virulence on this host.However, when Mak+ and Mak- progeny were compared insimilar genetic backgrounds, Mak- progeny were alwaysless virulent (Fig. 2). Thus, no substitute for the contributionof metabolic detoxification toward enhancement of virulencehas yet been found. Some Mak- field isolates from chickpea(Table 1) initially appeared to be likely sources of suchsubstitutes, but these isolates proved to be poor pathogens inthe virulence assay; one explanation may be that they weresecondary colonizers of already diseased tissue. The rela-tively high virulence observed previously in the Mak-isolate T-126 made it a logical candidate in which to seekpathogenicity determinants not dependent on metabolic al-teration of host phytoalexins, but none among the six Mak-progeny examined from cross 156 had such traits. Therefore,the role of phytoalexin detoxification as a contributing factortoward the virulence of N. haematococca MP VI on chick-pea appears to be similar to that for pisatin detoxificationduring pathogenesis of this fungus on pea (10, 13, 17).Whether nondegradative tolerance also plays a part in the

virulence of MP VI on chickpea is, however, still unclear.For example, even though Mak- progeny from crosses 230,269, and 272 were less virulent than their Mak+ siblings,many produced larger lesions on chickpea than the Mak-field isolate T-126 (Fig. 2 and 3) and larger lesions on thishost than were ever observed for Pda- isolates on pea (10,13, 17, 19). Whether this derives from genes controllingnondegradative tolerance or from introduction of other vir-ulence factors from the virulent Mak+ parent cannot bedetermined at present. No segregation for nondegradativetolerance was detected in crosses, in spite of previouslynoted variation in this trait among field isolates (e.g., T-126is fairly tolerant of maackiain [11]); all Mak- progeny in thisstudy had a similar level of tolerance (20 to 40%). That noneof the progeny exhibited an extreme sensitivity like that ofMP I might be explained if the genes for nondegradativetolerance were identical in the original parents (so that therewas no segregation) or if there were many independentlysufficient loci for this phenotype (so that any progeny islikely to have at least one).The results of this study are wholly consistent with the

interpretation that phytoalexin detoxification plays a role inthe pathogenicity of N. haematococca MP VI on chickpea,but recent findings of chromosome instability in the fungusindicate that this must be a tentative conclusion. Karyotypeanalysis of N. haematococca MP VI by pulsed-field gelelectrophoresis has shown unusual meiotic behavior in achromosome thought to contain Makl that explains theaberrant segregation of this gene; this chromosome was

partially deleted or completely missing in the Mak- progenyof cross 272 examined (14a, 14b). If a Mak- parent had sucha deletion (preliminary results support this possibility), thenit would not be able to provide a homologous partner forpairing and meiotic recombination with a normal Makichromosome, and as a result, all genes on the Maki chro-mosome would be coinherited. Then maackiain metabolism

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814 MIAO AND VANETTEN

might appear to be responsible for increased virulence,because Maki was linked to other genes which mediate highvirulence. Further investigation of this possibility by con-ventional genetic methods would require identification ofMak- strains with a true Makl homolog, i.e., one with aninactive allele rather than a chromosomal deletion. WhileMak3 appears to be a conventional Mendelian locus, there isevidence that Mak2 may also be on an unstable chromosome(14a). An alternative way to resolve remaining uncertaintieswould be to individually disable, by DNA-mediated trans-formation and homologous gene replacement techniques,each Mak gene in a virulent strain and then evaluate changesin tolerance and virulence; such investigation should rigor-ously discriminate the effects of Mak genes from those ofgenes closely linked to the Mak loci.

ACKNOWLEDGMENTS

We acknowledge D. E. Matthews for review of the manuscriptand D. Funnell-Baerg for assistance on studies of field isolates.

This work was supported in part by Energy Bioscience grantDE-FGO2-89ER14038 from DOE. V.P.W.M. was partially sup-ported by a Postgraduate Fellowship from the Natural Science andResearch Council of Canada.

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