Organ specificity of fungal pathogens on host and non-host plants

Ptysiologiral

and Molecular

PIOIII Pathologv

Organ specificity host plants W.

SCHAFER*

* Ias/i!ul /iir f Depnrlmea!

( 1994)

of fungal

211

45, 2 I l-2 18

pathogens

on tiost and non-

and 0. C. YODER~

Geabiologische Forsrhung, Ihaestr. 63, 14195 Berlin, Germany of Plant Palhologv, Cornell Unioersi!v, Ithaca, NY 14853, U.S.A.

(drcepepledfor publicalioa

JNNC 1994)

Cochliobolus heterostrophus and Nertria haemalococca are fungal pathogens of maize and pea, respectively. C. heterostrophus infects the foliage, but not the roots of maize whereas N. haemafococca attacks the roots and the stem base, but not the leaves of pea. A recombinant strain of C. heterosfrophas overexpressing the PDA-79 gene, which encodes pisatin demethylase of N. haema~ococca, colonized unwounded pea leaves but not pea roots and thus retained its organ specificity on a non-host plant. JV. haemalococca did not infect unwounded leaves of its own host, pea. Under our experimental conditions, the organ specificity of C. heferostrophus on pea was unchanged after wounding the tissues, while N. haematococca rapidly colonized the aerial parts of pea after wounding.

INTRODUCTION Many fungal pathogens display organ specificity in that they do not normally attack all parts of their host plant. Two such fungi are Cochliobolusheterostrophus, a pathogen of the foliage but not the roots of maize (
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W. Shafer and 0. C. Yoder 212 (obtained from H. D. VanEtten) contains one active pisatindemethylase (PDA) locus, locus PDAI [3, 71 and was propagated as described by VanEtten & Barz [I.!?]. Escherichinco/i K-12 strain DH5cr was usedfor propagation of the vector pUP1, which carries the PDA-encoding gene PDA-‘Z-9 and vector pUCH1, which is the same but without PDA-T9. Both plasmidshave been described previously [9 1. Fungal DNA was isolated as described [12). Restriction enzyme digestions, plasmid DNA preparations and southern analysis were carried out according to standard protocols [8].

Pathog&ci(y testson roots Pot assayswere performed according to VanEtten [14] with the modification that the autoclaved soil was infested with conidia immediately after sowing pea seedsof cv. Alaska 2B and the number ofconidia was reduced to a total of IO’ per 11 cm pot.‘Plants were incubated at 28 “C in a controlled environment chamber. Diseasedevelopment was evaluated 4 weeks after inoculation. Pathogeni+ testsonpea stems Peaswere grown in test tubes containing vermiculite and Hoagland’s solution in a high humidity growth chamber for 6 days [.Z,3, 161. Epicotyls v:ere inoculated with a 3 mm diameter cylinder of agar infested with mycelium. The chamber was sealed and resulting lesionswere evaluated after 6 days. Wounds were produced at the infection site by puncturing with a 26 gauge needle. Wounded control plants were inoculated with an agar cylinder without mycelium; they showed no or very little (< 1 mm) discoloration. Pathogenic+ testson leaves Peaswere grown as described for the pea stem assay, except that they were 8 days old when inoculated. Each leaf was inoculated with one drop of conidial suspension(1000 conidia in 15 pl water). Plants were incubated in 100% humidity in sealed chambers and lesions were evaluated after 6 days. Wounds in the leaves were produced by puncturing with a 27 gauge needle prior to inoculation. Lesion development was assessed after 3 days. Control plants were inoculated with 15 pl water without conidia. Wounded control leaves showed no discoloration around the wounding site. RESULTS

Construction of the PDA overproducing transformant C2-P and the vector control transformant C2-V was described earlier [9]. Southern blot analysis showed that a single recombination event between pUP1 and its homologous sequence (promoter 1) in the C. heterostrophusgenome produced a recombinant chromosome carrying approx. 18 copies of pUP1, arranged head-to-tail in tandem and flanked by direct repeats of the target sequence (Figs 1 and 2). C2-V carried 11 copies of the control plasmid pUCH1 [II], integrated by homology and arranged in a head-to-tail fashion. Transformant C2-P produced approx. twice as much PDA as did wild type .M haematococcu strain 77-2-3 [9]. To evaluate the impact of the expression of PDA-T9 gene in C. heterostrophus, leaves on intact pea plants were inoculated with equal amounts of conidia from strains CZ-

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FIG. I. Southern blot analysis of genomic DNAs from transgenic and wild type strains of Cochliobolus heleroslrophus and fledrio haemalococco. (a) DNA samples of N. haematococca strain 77-23, C. he/eros~roplws strains C2-P, C2-V and C2 (lanes l-4) were digested with StuI (which has no site in the vector). Digested DNAs were fractionated on an agarose gel, transferred to membrane and probed with the vector. Bands in lanes I and 4 represent single copies of PDAI (in JV. haematococca) and promoter I (in C. heterostrophus; see [13]), respectively. Heavy bands in lanes 2 and 3 reflect multiple copiesofpUP1 (in C2-P) and pUCHl (in C2-V), respectively. (b) The same DNA samples as in (a) were digested with XbaI (lanes l-4), which has one site in the vector, or with XhoI (lanes 5-8), which has no site in the vector. Digested DNAs were fractionated on an agarose gel, transferred to a membrane and probed with the 3.35 kb fragment carrying PDA-79 [9]. The band in lanes I and 5 represents the single copy of PD.41 in JK haemafococca. The heavy band in lanes 2 and 4 indicates multiple tandemly repeated copies of pUPl in the genome of C2P; lighter bands in lane 2 represent the border fragments. Lanes 3,4,7 and 8 have no bands, since control strains C2-V and C2 do not have PDA-79 in their genomes. Molecular size markers are given in kb.

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P, C2-V or the N. haematococca control strain 77-2-3. After 6 days, 77-2-3 inoculated pea leaves showed no macroscopic symptoms, C2-V inoculated leaves possessedtiny necrotic flecks and slight chlorosis, while C2-P inoculated leaves had developed large lesions (Fig. 3). This experiment was performed four times, with 15 inoculated leaves per isolate each time. All experiments gave the results shown (Fig. 3). Moreover, when leaves bearing lesions were placed in a moist chamber for 3-4 days, the fungus sporulated and hyphae were microscopically visible growing among the leaf cells. When pea leaves were puncture wounded before inoculation, the C. heterostrophus transformants (C2-V and C2-P) caused symptoms indistinguishable from those shown in Fig. 3. for unwounded leaves. N. haematococca, -however, invaded and rapidly colonized wounded leaves (Fig. 4). In another set of experiments, lesion development on unwounded stems was tested (Fig. 5). C2-V caused no symptoms on any of the 10 replicate stems, 77-2-3 caused no lesionson nine of 10 replicate stems (on one stem a large lesion developed), and C2-P caused a small lesion on each of the 10 replicate stems. Under the conditions of the experiment, 77-2-3 was either unable to infect intact pea stems or did so inconsistently.

W. Shafer and 0. C. Yoder

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C. hetemstmphus genomic DNA

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C. hetemstmphus

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FIG. 2. Integration of pUP1 into the genome of Cochliobolas heferostrophns by homologous recombination at the promoter I site (above). Copy number of the inserted vector in the recombinant chromosome was estimated by dot-blot analysis [9] to be 18 (below). PD.4 is under the control of its native promoter.

FIG. 3. Symptoms on pea leaves caused by transgenic Cochliobolw hcferostrophus and by Necfria haematococca on pea leaves. Leaves of 8-day-old pea plants were each incubated conidia in 15 pl of water and checked for symptoms after 6 days incubation in a moist From left to right: 77-2-3 (N. haematococca), C2-V and C2-P. C. heterostrophus wild type produced the same symptoms as strain C2-V.

wild type with 1000 chamber. strain C2

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FIG. 4. Effect of wounding on the colonization of pea leaves by Neco% hacmntococcu. Leaves of 8-day-old pea plants were either left unwounded (centre), or puncture wounded with a needle (right) and inoculated with 1000 conidia in 15 pl of water. Symptoms were checked after 4 days incubation in a high humidity chamber. Controls were wounded leaves inqculated with 15 pl water (left).

FIG. 5. Results of inoculating non-wounded pea stems with Cochliobolus Each stem was inoculated with mycelium and incubated chamber. There were 10 replicate plants per treatment. Each treatment three times. From left to right: C2-V, C2-P and 77-2-3.

haematococca.

or Necfriu for 6 days in a moist was repeated at least

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Since JV. haematococca is a soil inhabiting fungus its points of attack are thought to be the cotyledon attachment area, the underground epicotyl, and the upper tap root. To evaluate pathogenicity with an assay that resemblesthe natural situation, pea seeds were planted in sterilized soil inoculated with 10’ conidia per pot [14] and disease development was evaluated 4 weekslater. Forty percent of the plants in soil containing 77-2-3 were dead within 3 weeks while the rest were stunted. Roots of all plants were necrotic (Fig. 6). Strains C2-v and C2-P had no observable effect on pea roots. Thus

FIG. 6. Results of growing pea plants in fungus-infested soil. Pea plants were grown for 4 weeks in soil inoculated with conidia (IO’ per pot). At least 20 plants were treated with each fungal strain. From left to right: C2-V, C2-P and 77-2-3.

C. heterostrophur cannot attack pea roots even if it produces twice as much PDA [9] as the wild type of the very effective root pathogen .N. haematococca strain 77-2-3. DISCUSSION

Our results show that C. heterostrophw, a natural pathogen of the foliage but not the roots of maize, maintains its organ specificity on a non-host, pea, which it can colonize when transformed to overproduce the phytoalexin detoxifying enzyme PDA. Both wounded and intact pea leaf tissueswere attacked by transgenic C. heterostrophus. In contrast, under our experimental conditions, N. haematococca, a pathogen of pea roots and stem bases,did not attack the leaves ofits own host plant aslong asthey were intact (resultswith stemswere variable). Wounded pea leaves or stemswere rapidly colonized by N. haematococca, suggestingthat the epidermis may play a role in the organ specificity of this fungus. The molecular basisof fungal organ specificity is not known. However, the work of Keller et al. [I, IO] h as associated the pH optimum of fungal cutinase with organ

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specificity. The general observation is that foliage pathogens tend to produce cutinase with an acidic pH optimum whereas root pathogens usually produce cutinase with an alkaline pH optimum. The addition of cutinase with an acidic pH optimum to the inoculum of a non-leaf invading fungal strain, which itself produced cutinase with an alkaline pH optimum, enabled the fungus to attack leaves [IO]. Our observations on the organ specificity of C. heterostrojjhus and JV. haematococca are consistent with those of Keller et al. C. heterostrophus produces cutinase with an acidic pH optimum [IO] and colonizes aerial plant parts only, whether it is on its own host (corn) or a non-host (pea). N. haematococca produces cutinase with an alkaline pH optimum and colonizes roots and stem basesonly. This association between cutinase pH optimum and organ specificity predicts that if N. haematococca were constructed to produce cutinase with an acidic pH optimum it would be able to attack unwounded leaves, and that if C. heterostrophus were constructed to produce cutinase with an alkaline pH optimum it would be able to attack roots. In an attempt to test the second prediction, C. heterostrophus transformants have been constructed with produce loo-fold more .N. haematococca cutinase (which has an alkaline pH optimum) than native cutinase (Oeser & Yoder, unpublished data). The expectation was that the transformants would be able to attack roots. However, these transformants were not only unable to colonize roots of maize (the natural host of C. heterostrophus), they were also unable to invade roots of the non-host pea, even if they were transformed to tolerance of the pea phytoalexin pisatin. Thus, although cutinase pH optimum may play a role in fungal organ specificity, we have not yet been able to demonstrate it. This work was supported by a fellowship to W. Schafer by the Deutsche Forschungsgemeinschaft and by grants from the U.S. Department of Agriculture, the Environmental Protection Agency and the Cornell Biotechnology Program.

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2. 3. 4. 5.

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WE, Yoder OC, Apple AR. 1984. Influence of naturally occurring marker genes on the ability of Cochliobolus helerostrophus to induce field epidemics of southern corn leaf blight. P&ytopathology 74: 175-178. Hoagland DR, Arnon DI. 1938. The water culture method for growing plants without soil. University of California Agricultural Experimen! Slation Circular Jfo. 97, p. 39. Kistler HC, VanEtten HD. 1984. Regulation of pisatin demethylation in Nectria haemafococcu and its influence on pisatin tolerance and virulence. Journal of General Microbiology 130: 2605-2613. Kiiller W, Parker MD. 1989. Purification and characterization of cutinase from Venturia inaequafis. Phytopalhology 79: 278-283. Kraft JM, Burke DM, Haglund WA. 1981. Fusarium diseases of beans, peas and lentils. In: Nelson PE, Toussoun TA, Cook RJ, eds. Fusarium: Diseases, Biology and Taxonomy. The Pennsylvania State University Press, 142-157. Leach J, Lang BR, Yoder OC. 1982. Methods for selection ofniutants and in u&o culture of Cochliobolus heleroslrophus. journal of General Microbiology 128 : 17 19-l 729. Miao VPW, Matthews DE, VanEtten HD. 1991. Identification and chromosomal locations of a family of cytochrome P-450 genes for pisatin detoxification in the fungus Neclria haemafococca. Molecular and General Genetics 226 : 2 14-223. Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular Cloning: A Laboratory Manual. New York: Cold Spring Harbor Laboratory Press. Schafer W, Straney D, Ciuffetti L, VanEtten HD, Hoder OC. 1989. One enzyme makes a fungal pathogen, but not a saprophyte, virulent on a new host plant. Science 246: 247-249. 15

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10. Trail F, Keller W. 1990. Diversity ofcutinases from plant pathogenic fungi: Evidence for a relationship between enzyme properties and tissue specificity. Physiological and Molecular Plant Pathology 36: . 495-508. 11. Turgeon BG, Bohlmann H, Ciuffetti LM, Christiansen SK, Yang G, Sch&fer W, Yoder OC. 1993. Cloning and analysis of the mating type genes from Cochliobolus heterostrophus. Molecular and General Genetics 238: 270-284. 12. Turgeon BG, Garber RC, Yoder OC. 1985. Transformation of the fungal maize pathogen Cochliobolus heterostrophus using the Aspergillus nidulans amdS gene. Molecular and General Genetics 201: 450-453. 13. Turgeon BG, Garber RC, Yoder OC. 1987. Development of a fungal transformation system based on selection of sequences with promoter activity. Molecular and Cellular Biology 7: 3297-3305. 14. VanEtten HD. 1978. Identification of additional habitats @fNectria haematococca mating population VI. Phytopathology 68: 1552-1556. 15. VanEtten HD, Barz W. 1981. Expression of pisatin demethylation ability in Nectria haematococca. Archives of Microbiology 129 : 56-60. 16. VanFkten HD, Matthews PS, Tegtmeier KJ, Dietert MF, Stein JI. 1980. The association of pisatin tolerance and demethylation with virulence on pea in Nectria haematococca, Physiological aud Molecular Plant Pathology 16: 257-268.