CURRENT MICROBIOLOGY Vol. 50 (2005), pp. 319–323 DOI: 10.1007/s00284-005-4509-6

Current Microbiology An International Journal ª Springer Science+Business Media, Inc. 2005

Biological and Molecular Variability of Sarocladium oryzae, the Sheath Rot Pathogen of Rice (Oryza sativa L.) Niraikulam Ayyadurai, Sundar Isaac Kirubakaran, Sirobhooshanam Srisha, Natarajan Sakthivel Department of Biotechnology, Pondicherry University, Kalapet, Pondicherry 605014, India Received: 10 November 2004 / Accepted: 2 January 2005

Abstract. Sheath rot disease of rice caused by Sarocladium oryzae (Sawada) (=Acrocylindrium oryzae, Sawada) has become an important production constraint in all rice-growing countries. Pathogenicity, phytotoxic metabolites, and random amplified polymorphic DNA (RAPD) markers were used to assess the level of genetic variability of S. oryzae derived from rice cultivars, CR1018, IR36, and IR50, of different locations in North East and South India. Variability in pathogenicity, phytotoxic metabolite production, and DNA polymorphisms was detected among S. oryzae isolates. Results indicated that S. oryzae isolates produced both cerulenin and helvolic acid at concentrations 0.3–0.62 and 0.9–4.8 lg mL)1 of culture filtrate, respectively. Isolates that produce higher concentration of helvolic acid induced a high percent incidence of sheath rot disease. Oligonucleotide primers, GF and MR, generated either a simple (up to 2 bands) or complex (up to 6 bands) RAPD pattern. According to their level of similarity, S. oryzae isolates from North East and South India were grouped separately into two major clusters and 13 genotypes. Molecular- and pathogenicity-based classifications were not correlated, but a high level of genetic variability within S. oryzae isolates was identified. The molecular variability of S. oryzae isolates will be an important consideration in breeding programs to develop durable resistance for sheath rot disease.

Sarocladium oryzae (Sawada) W. Gams and D. Hawksworth, (=Acrocylindrium oryzae, Sawada) [11] causes sheath rot disease of rice (Oryza sativa L.). Due to the introduction of high-yielding and semi-dwarf rice cultivars, S. oryzae has become a hindrance to rice production in all rice-growing countries. The disease can cause yield losses of 3–85% depending upon the disease severity [2, 6]. All popular rice cultivars are highly susceptible to S. oryzae [2]. The fungus produces characteristic grayish-brown lesions on the uppermost flag leaf enclosing the panicle. Severe infection has been reported to produce partially emerged or totally compressed panicles with chaffy grains [2, 7, 8]. S. oryzae-infection reduces the seed viability and nutritional value. Although S. oryzae is seed-borne and seed transmitted, the fungus also survives as mycelium in infected plant residues, weed hosts, and soil [6, 8].

Correspondence to: N. Sakthivel; email: natarajansakthivel@yahoo. com

Wind, stem-borer, mites, and mealy bugs disseminate the conidia [2, 19]. We have recently described the disease cycle and epidemiology of S. oryzae [19] and also reported the production of phytotoxic metabolites in culture [20] and in infected grains [12]. Seed treatments with fungicide do not kill the fungi present inside the glumes [19] and, therefore, fungicide treatments have been unsuccessful in S. oryzae control or have proved too expensive as well harmful to the environment. Biological control of S. oryzae has been limited due to the inconsistency of antagonists in field conditions [18]. Therefore, the development of resistant cultivars deserves attention. Successful breeding and effective deployment of durable plant resistance require an understanding of pathogen diversity and of the way in which virulence evolves in the pathogen population [14]. Random amplified polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP) or genetic fingerprinting approaches were used to analyze the population structure of plant pathogenic fungi [9, 15]. RAPD or arbitrarily primed-PCR (AP-PCR)-based

320 methods are most suitable with respect to speed of processing, although the selection of primers and amplification conditions may involve considerable initial work [5]. To the best of our knowledge, there is no report concerning the molecular variability of S. oryzae. In the present work, molecular variability among S. oryzae isolates derived from North East and South India has been studied in terms of pathogenicity, phytotoxic metabolite production, and DNA polymorphism.

CURRENT MICROBIOLOGY Vol. 50 (2005) Table 1. Geographic location, cultivar origin, and RAPD pattern of Sarocladium oryzae by GF and MR primers RAPD pattern (S = simple; C = complex) Geographic location

Rice cultivar

S. oryzae isolate

GF

MR

North East India West Bengal

CR1018

SO1 SO2 SO3 SO4 SO5 SO6 SO7 SO8 SO9 SO10 SO11

S S S S S S S S S S S

S S S S S S S S S S S

South India Tamilnadu

IR36

SO12 SO13 SO14 SO15 SO16 SO17 SO18 SO19 SO20 SO21 SO22 SO23 SO24 SO25 SO26 SO27 SO28 SO29 SO30 SO31 SO32

C C C C C C C C C C S S S S S S S S S S S

C C C C C C C C C C C C C C C C C C C C C

Materials and Methods Microbial cultures, plant materials and chemicals. Citrobacter michiganensis (Department of Plant Pathology Culture Collection, Kansas State University, Manhattan, KS; no.1150) and Candida albicans (ATCC2140) were supplied by Bruce Raymundo (Department of Plant Pathology, Kansas State University) and Susan Porter (Clinical Laboratory of Peterson, Manhattan, KS), respectively, and used in selective bioassays for the estimation of metabolites. Susceptible rice cultivar IR36 was used for pathogenicity testing. Authentic samples of cerulenin and helvolic acid were purchased from Sigma (St. Louis, MO). All solvents and reagents used were analytical or liquid chromatographic-grade chemicals. The isolates of S. oryzae are maintained in the Department of Biotechnology, Pondicherry University. Collection of samples and isolation of fungi. A survey was made in the rice-growing areas of North East and South India for the occurrence of sheath rot. The surveyed states and rice cultivars are shown in Table 1. One infected sheath was collected from each site. The fungus was isolated from infected sheath tissue following the method described previously [2, 18]. Identification of S. oryzae was done on the basis of cultural characteristics and spore morphology as described earlier [2, 11]. Single-spore cultures were maintained at 30
C on potato dextrose agar (PDA) or on yeast glucose agar (YEG) both of which were made acidic (pH 4.0) using 0.1N HCl. For longterm storage, S. oryzae mycelia grown on PDA were scraped, resuspended in 25% glycerol, and stored at )80
C [1]. Pathogenicity test. To test pathogenicity, rice plants of cultivar IR36 were grown in the greenhouse. At booting, the panicle-emerging stage, tillers were inoculated with S. oryzae following the standard grain inoculum technique [18]. A total of 100 tillers from 20 plants were inoculated with each isolate of S. oryzae. The incidence of sheath rot disease was recorded after 14 days using the standard evaluation system for rice (International Rice Research Institute, Philippines, 1988). The results were subjected to analysis of variance and DuncanÕs Multiple Range Test (DMRT). Extraction, purification, and quantification of phytotoxic metabolites, helvolic acid, and cerulenin. Extraction, purification, and quantification of metabolites produced by S. oryzae were carried out following standard procedures [12]. Briefly, S. oryzae cultures were grown in 200 mL of liquid medium containing 6% glucose, 0.5% peptone, 0.30% NaCl, 0.30% CaCO3 for 2 days at 30
C. The culture medium was filtered to remove mycelium, extracted with an equal volume of chloroform, dried, and dissolved in 1 mL of ethanol and purification of the extract was done by silica gel column. The amount of helvolic acid and cerulenin was determined by selective bioassays, employing C. michiganensis or C. albicans following the method described previously [12] and the results were subjected to analysis of variance and DMRT.

Pondicherry

IR50

Extraction and quantification of fungal DNA. Genomic DNA was extracted from the mycelia of S. oryzae following the cetyltrimethylammonium bromide (CTAB) method [10]. Isolates of S. oryzae were grown in YEG medium under continuous shaking for a period of 7 days. Mycelia after centrifugation at 3,000g for 10 min at 4
C were frozen in liquid nitrogen and lyophilized. The powdered mycelia were suspended in CTAB extraction buffer incubated at 65
C for 45 min and centrifuged. The supernatant was removed and mixed with an equal volume of phenol:chloroform (1:1, vol/vol) and centrifuged. The top layer was removed and to this a mixture of choloroform:iso-amyl alcohol (24:1, vol/vol) was added. After centrifugation, DNA was precipitated using ice-cold ethanol. Purity and the concentration of DNA were estimated in a U2000 spectrophotometer (Hitachi Ltd., Tokyo, Japan). PCR amplification and agarose gel electrophoresis. Oligonucleotide primers, RY, 5¢(CAG)53¢; GF, 5¢(TCC)53¢;

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N. Ayyadurai et al.: Biological and Molecular Variability of S. oryzae CAT, 5¢ (CAT)53¢; MR, 5¢GAGGGTGGCGGTTCT3¢ that produce RAPD in filamentous fungi [5] were used in this study. Primers were synthesized from Integrated DNA Technologies Inc., Coralville, IA. PCR reaction (50 lL) contained 50 ng of primer, 100 ng of fungal DNA, 1· Taq DNA polymerase buffer, 0.025 units of Taq DNA polymerase, 0.2 mM of each deoxynucleotide triphosphate, and 1.5 mM MgCl2. Amplification was performed in a DNA thermal cycler (2400 cycler, Perkin Elmer International, Rotkreuz, Switzerland) programmed for an initial denaturation cycle (95
C for 30 s) followed by 30 cycles of 1 min at 95
C, 1 min at 42
C, and 1 min at 72
C with an extension of 72
C for 10 min. RAPD products were size fractionated on a 0.8% agarose gel (Promega, Madison, WI) with 0.3 lg mL)1 ethidium bromide using 0.5· TBE (45 mM Tris-borate, 1 mM EDTA, pH 8.0) running buffer at a constant voltage of 5 V cm)1. A 1-kb ladder DNA (Bethesda Research Laboratory, Bethesda, MD) was included as molecular weight marker. The gels were visualized on an ultraviolet transilluminator. RAPD analyses. DNA banding profiles were analyzed using the Quantity One program (Bio-Rad Laboratories, Richmond, CA). For data analysis, each amplified fragment of different molecular size was treated as a separate character. DNA fragments of the same size were assumed to represent homologous allele and scored as either present or absent. Similarities were determined from the similarity coefficient (Dice) and clustering was by the unweighted pair group method arithmetic (UPGMA) algorithm. Calculations were undertaken through the NTSYS pc2 package (Exeter Software, State University of New York, Stony Brook, NY).

Results and Discussion All isolates were identified as S. oryzae based on morphological characteristics (white cottony aerial mycelium, irregularly branched conidiophores with one or two whorls of appressed branches and fusiform, curved, hyaline, single-celled conidia). Two weeks after inoculation, S. oryzae-inoculated tillers of rice plants (IR36) produced typical sheath rot lesions on the uppermost flag leaf enclosing the panicle. As shown in Table 2, S. oryzae isolates produced disease incidence of 45–98%. S. oryzae is known to produce phytotoxic metabolites such as helvolic acid and cerulenin. Helvolic acid is reported to interfere with chlorophyll biosynthesis [4] and cerulenin inhibits methyl salicylic acid [16] and fatty acid metabolism [17]. Since both metabolites are phytotoxic as well as antimicrobial, S. oryzae is antagonistic to other microorganisms and exhibits more pathogenic potential [13]. All the S. oryzae isolates used in this study produced cerulenin as well helvolic acid at concentrations 0.3–0.62 and 0.9–4.8 lg mL)1 of culture filtrate. Isolates that produce a higher concentration of helvolic acid induced a high percent incidence of sheath rot disease (Table 2). However, several other mechanisms such as cerulenin, pectic and cellulolytic enzymes could have contributed also to the aggressiveness of S. oryzae [21]. Primers such as RY and CAT did not produce amplification possibly due to scattering of the RY and

Table 2. Pathogenicity and production of phytotoxic metabolites, helvolic acid, and cerulenin by Sarocladium oryzae isolates Metabolites (lg ml)1) S. oryzae isolate

Pathogenicity (% incidence)

Helvolic acid

Cerulenin

SO1 SO2 SO3 SO4 SO5 SO6 SO7 SO8 SO9 SO10 SO11 SO12 SO13 SO14 SO15 SO16 SO17 SO18 SO19 SO20 SO21 SO22 SO23 SO24 SO25 SO26 SO27 SO28 SO29 SO30 SO31 SO32

64bc 45a 59bc 61bc 95c 85de 79d 97fg 91ef 64bc 90e 84de 92ef 79d 93ef 98g 52b 85dc 60bc 97fg 90e 85de 50ab 79d 95e 80de 95e 91ef 98g 78cd 90e 65c

3.1bc 1.0a 0.9a 3.2c 4.5de 3.7cd 1.9ab 4.8e 4.4d 2.5bc 2.9bc 1.2a 2.8bc 3.9cd 4.7dc 3.8cd 1.9ab 2.2b 2.8bc 3.5c 3.9cd 3.3c 2.9bc 2.4b 1.2a 1.0a 4.5de 4.5de 4.8e 3.0bc 1.8ab 0.9a

0.3a 0.35a 0.5c 0.38ab 0.4ab 0.42b 0.3a 0.4ab 0.3a 0.52c 0.5c 0.46bc 0.42b 0.6cd 0.3a 0.63d 0.5c 0.63d 0.48bc 0.55c 0.4ab 0.55c 0.56cd 0.52c 0.3a 0.43b 0.62d 0.62d 0.5c 0.42b 0.32a 0.3a

Note. Each value is an average of 3 replicates. Means within the column followed by different letters are significantly different (P = 0.05) by DuncanÕs Multiple Range Test (DMRT).

CAT sequences in the genome of S. oryzae [5]. From the initial screening, GF and MR primers were selected on the basis of reproducible and polymorphic patterns. Both of these primers generated either a simple (up to 2 bands) or complex (up to 6 bands) RAPD patterns under optimized PCR conditions (Table 1, Fig. 1). The level of similarity among the MR and GF primers ranged from 0.52–1, indicating a high degree of polymorphism within S. oryzae. All S. oryzae isolates were divided into two major clusters and 13 genotypes. The first major cluster was further grouped into two subgroups with the level of similarity at least equal to 0.67. The first major cluster consisted of mostly North East Indian (West Bengal) isolates represented by CR1018 as well two

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CURRENT MICROBIOLOGY Vol. 50 (2005)

Fig. 1. RAPD band profiles of single-spore isolates of Sarocladium oryzae detected by the computer using Quantity One Program (Bio-Rad Laboratories, Richmond, CA). RAPD band profiles generated by (a) GF primer and (b) MR primer. Lane 1, 1-kb ladder DNA; Lanes 2–33, S. oryzae isolates SO1–SO32.

Fig. 2. Dendrogram of single-spore isolates of Sarocladium oryzae showing two major clusters and 13 genotypes. Dendrogram obtained from similarity coefficient (Dice) after UPGMA clustering of RAPD band combined data generated by MR and GF primers.

South Indian (Pondicherry) isolates represented by IR50. The second major cluster consisted of South Indian isolates represented by IR36 and IR50 with the level of similarity at least equal to 0.69. The two Pondicherry isolates, SO22 and SO32, are most distinct from the others and formed a separate group (Fig. 2). All modern rice cultivars are highly susceptible to S. oryzae. Such susceptibility of the most popular rice cultivars negates the possibility of a lineage-based pathogen-control strategy. If pathogens are specialized on one of the host genotypes, then transferring resistant genes between gene pools may provide a more durable

resistance [3]. Due to the high degree of DNA polymorphism, lack of correlation between virulence and DNA pattern has been reported in plant pathogenic fungi such as P. brassicae [14] and Puccinia striiformis [7]. In this study, S. oryzae isolates collected from North East and South India were analyzed. All the isolates showed a high variability in pathogenicity, phytotoxic metabolite production, and DNA pattern. As evidenced in other plant pathogenic fungi [7, 14], there was a lack of correlation between pathogenicity and the RAPD pattern of S. oryzae may be due to the high degree of DNA polymorphism. Results revealed that the North East iso-

N. Ayyadurai et al.: Biological and Molecular Variability of S. oryzae

lates and South Indian isolates were independently grouped into two major clusters and further subdivided into different genotypes. All the S. oryzae isolates (except SO10) that originated from the West Bengal state of North East India specialized in the rice cultivar, CR1018, and clustered as a separate genotype. This study can be considered as a first report on the biological and molecular variability of S. oryzae due to the absence of any report in the literature. Understanding the molecular variability of S. oryzae may be valuable in designing breeding programs to develop durable resistance. ACKNOWLEDGMENTS We thank Bruce Raymundo, Department of Plant Pathology, Kansas State University, Kansas, and Susan Porter, PetersonÕs Clinical Laboratory, Manhattan, Kansas, for supplying microbial cultures.

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