Cell & Plant Sci 2011 2 (3): 9-11

Journal of Cell & Plant Sciences

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Short Communication

Cultur e Media Influence on vegetative growth and In vitr o conidia production of

Magnapor the or yzae

Susan Partridge-Metz and Ambika Chandra*
Texas AgriLife Research and Extension Center, Texas A&M System, 17360 Coit Road, Dallas TX 75252

Received: 13.4.2011

Accepted: 07.05.2011

Published: 30.05.2011

Abstract A local isolate of Magnaporthe oryzae collected from St. Augustinegrass [Stenotaphrum secundatum (Walt.) Kuntze] was used to compare the influence of five culture media on the vegetative mycelial growth, pigmented mycelial production and conidia production of M. Oryzae eleven days after incubation at 26 C under diurnal (12hr) fluorescent light. The five culture media included in the present study are potato dextrose agar (PDA), oatmeal agar (OMA), V8 agar (V8A), prune juice agar (PJA) and St. Augustinegrass agar (STAA). Vegetative mycelial growth was highest using V8A, PDA and OMA, while the culture media producing the highest amount of pigmented mycelia were OMA, STAA and V8A. The highest yield of conidia was obtained using STAA, which was verified in two separate experiments.
Key words:, Conidiophore, Pyricularia oryzae, Pyricularia grisea, St. Augustinegrass

Corresponding Author: A. Chandra, e-mail a-chandra@tamu.edu, Phone: + 9722315362, Fax: +9729529216

INTRODUCTION The ascomycete fungus Magnaporthe oryzae (Couch) [anamorph = Pyricularia oryzae Cav.] formally known as Magnaporthe grisea (Herbert) [anamoph = Pyricularia grisea (Cooke) Sacc.] Couch and Kohn (2002), produces gray conidiophores which yield terminal pyriform asexual spores termed conidia, known to cause infection in more that 50 monocot species (Ou 1980). M. oryzae causes gray leaf spot disease in St. Augustinegrass [Stenotaphrum secundatum (Waltz.) Kuntze] and perennial ryegrass [Lolium perenne (L.)] (Smiley et al. 1996), and rice blast disease in rice [Oryza sativa (L.)] (Yorionori and Thurston 1974). Large scale production of fungal spores of M. oryzae is crucial when making spore suspensions for disease screening studies. Several culture media have been reported to support conidia production of M. oryzae (Dhingra and Sinclair 1995). Many researchers have used natural substrates in culture media to increase spore production and maintain isolate virulence after successive fungal transfers (Snyder 1947; Yorionori et al. 1974; Bhattacharyya and Bose 1981; Satyanarayana and Reddy 1985; Vincelli and Dixon 2002). The purpose of this study was to quantify and compare vegetative mycelial colonization, pigmented mycelial production and condia production of a local isolate of M. oryzae on following culture media substrates; natural (STAA), dehydrated (PDA and OMA), juice extract (PJA) and juice concentrate (V8A).

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Cell & Plant Sci 2011 2 (3): 9-11 MATERIALS AND METHODS Leaf tissue of St. Augustinegrass exhibiting lesions identified as gray leaf spot disease (Smiley et al. 1996) was excised from plant material, surface sterilized in ethyl alcohol 950 mL L-1 for 15 sec., followed by sodium hypochorite 100 mL L-1 for 3 min., and again in ethyl alcohol 950 mL L-1 for 15 sec. Sterilized leaf tissue was blotted dry using sterile paper towels and placed on water agar containing 50 mg L-1 streptomycin, 50 mg L-1 tetracycline, and 50 mg L-1 chloramphenicol (Romero et al. 2001; Tredway et al. 2003). Hyphal tips (5mm plug) of this local strain of M. oryzae were transferred from water agar to five different culture media. The five culture media used in the present study are potato dextrose agar (PDA; 10 g dehydrated potato infusion Diffco L-1), oatmeal agar (OMA; 40 g dehydrated oatmeal infusion Diffco L-1), vegetable juice agar (200 mL V8 concentrated vegetable juice, 3 g CaCO3, 15 g agar L-1), prune juice agar (PJA; 200 mL water extract from dried prunes Sunshine 3 g CaCO3, 15 g agar L-1) and St. Augustinegrass agar (STAA; 40 g St. Augustinegrass leaf tissue, 5 mm-15 mm tissue length, 3 g CaCO3, 20 g sucrose, 15 g agar L-1). Vegetative mycelial colonization, percent pigmented mycelia and conidia production was determined in experiment one, and in experiment two, only conidia production was measured. In the first experiment, the STAA contained St. Augustinegrass tissue cut into a length

Metz & Chandra

of 15 mm, while the second experiment used amended water agar with St. Augustinegrass tissue cut into smaller lengths of 5mm. Six replicate Petri plates were used in experiment one and four replicate plates were used in experiment two. Petri plates of each culture media containing the fungal isolate were incubated for four days under dark conditions at 26 C and then exposed to a 12 h photoperiod for seven days. Pigmented mycelia has been reported to yield greater conidiophores and conidia as compared to the white vegetative mycelia (Latterell and Rossie 1986) thus, pigmented mycelia was measured to estimate the conidiophore production in this study. Pigmented mycelia (conidiophore production) were determined by measuring the area (mm2) of pigmented mycelia using a digital caliper for each replicate Petri plate. The condia spores were harvested at 11 days post transfer in both experiments by adding a 10 mL aliquot of sterile deionized water (pH 7.5) per 9 cm Petri plate and gently scraping the surface. A spore suspension from each media was prepared and spore densities were counted (six subsamples/suspension) using a hemacytometer. Data was subjected to analysis of variance (ANOVA) using SAS (2007) both within and between experiment one and two with Fishers protected least significant difference (LSD) used to compare the means for all of the variables within experiment one and experiement two (alpha = 0.05).

Table 1 Comparison of vegetative growth, vegetative pigmentation and conidia production of M. oryzae on five different culture media

Media a V8A PDA OMA STAA PJA P > value r2

a

Nb 6 6 6 6 6

Mycelial colonization (mm2) 4779.6 a 4543.0 a 4533.2 a 3742.1 b 2573.6 c < 0.0001 0.84

Experiment 1 (Mean)c Pigmented mycelia (mm2) Conidia/mL 3495.4 a 3.1 x 104 c 1727.4 b 1.0 x 104 c 4238.7 a 1.1 x 105 b 3728.6 a 1.5 x 105 a 1441.8 b 3.3 x 104 c < 0.0001 < 0.0001 0.82 0.82

Experiment 2 (Mean)c Nb 4 4 4 4 4

Conidia/mL 1.9 x 105 b 1.3 x 104 d 1.4 x 105 c 3.3 x 105 a 2.6 x 104 d < 0.0001 0.87

Culture media used to grow Magnaporthe oryzae; V8A=vegetable juice agar, PDA=potato dextrose agar,OMA=oatmeal agar,STAA=St. Augustingegrass agar, PJA=prune juice agar. b N of six = six replicate Petri plates measured per variable. N of four = four replicate Petri plates measured per variable. c Means followed by same letter in each column not significantly different according to Fishers protected least significant difference test.

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Cell & Plant Sci 2011 2 (3): 9-11 RESULTS AND DISCUSSION The local isolate of M. oryzae used in the present study produced both vegetative and pigmented mycelia on all five culture media. Significant differences were observed between the five culture media for vegetative mycelial colonization, pigmented mycelial production and conidia production (Table 1). Total vegetative mycelial colonization ranged from 4779.6 mm2 for V8A to 2573.6 mm2 for PJA. There was no significant difference between vegetative mycelial colonization using OMA (4533.2 mm2) or PDA (4543.0 mm2), both of which are commercially available dehydrated media. Vegetative mycelium on STAA (3742.1 mm2), water agar amended with St. Augustinegrass tissue 15mm length , colonized the Petri plate less than either PDA or OMA (p < 0.0001; r2 = 0.84). Vegetative mycelial colonization is not indicative of conidiophore production however, pigmented mycelial colonization provides an estimate of conidiophore production (Latterell and Rossie 1986). Using the STAA, a 3728 mm2 area or 99.6% of total mycelia colonizing the Petri plate produced pigmented mycelia and was statistically grouped with OMA and V8A where 93.0% (4238.7 mm2) and 73.1% (3495.4 mm2) of total colonized plate, respectively, produced pigmentation (conidiophores). The PDA produced significantly lower (38.0%; 1727.4 mm2) coverage of pigmented mycelia per plate (p < 0.0001; r2 = 0.82) and was statistically grouped with PJA (56%; 1441.8 mm2) (Table 1). Two separate experiments were conducted to estimate conidia production in five different media types. The results indicate that, STAA amended with St. Augustinegrass tissue ( 15mm or 5mm tissue length) yielded the highest numbers of conidia per mL in both experiments (1.5 x 105 conidia mL-1 and 3.2 x 105 conidia mL-1, respectively) as compared to other culture media tested. The higher conidia yield in the second experiment using STAA is likely to be due to smaller tissue size and better distribution of the substrate across the surface of the agar. Researchers have used water agar amended with perennial ryegrass tissue macerated in a blender (Vincelli and Dixon 2002). Although, not tested in the present study, we speculate that macerating St. Augustinegrass tissue prior to sterilization may yield an increase in conidia production using STAA. It was observed in the both experiments using the natural substrate (St. Augustinegrass tissue) that conidiophore production appeared more aerial in growth pattern as compared to other culture media. In the first experiment, a significantly higher conidia yield was produced using OMA (1.1 x 105 conidia mL-1) than either V8A (3.1 x 104 mL-1), PJA (3.3. x 104 mL-1) or PDA (1.0 x 104 mL-1). Latterell and Rossie (1986) reported that pigmented mycelium yielded more conidiophore and conidia production, when using a biphasic process with the

Metz & Chandra

pathogen grown first using inoculated steeped corn grown in shake culture, then dryed under clean conditions to produce a dry spore product. In the present study, pigmented mycelia developed to a lesser extent on PDA (38.0%) and PJA (56.0%) than V8A (73.1%) yet V8A, PDA and PJA were statistically grouped together when measuring conidia production, indicating that pigmented mycelia or conidiophore production may not necessarily translate into higher conidia production with M. oryzae grown on agar based media. In the second experiment, V8A (1.9 x 105 mL-1) yielded a significantly higher conidia production than OMA (1.4 x 105 mL-1). Conidia production on PJA and PDA was significantly less than using STAA, OMA and V8A. Previous research investigating the sporulation of P. oryzae, which causes the disease rice blast, Satyanarayana and Reddy (1985), stated that the use of rice nodes as the natural substrate in rice node dextrose agar (rice nodes plus 1% dextrose) produced a conidia yield of 8 x 104 mL-1, which was reported as an abundant level of conidia production. This communication reports using a natural, readily available substrate, St. Augustinegrass tissue, ( 15mm or 5mm tissue length) plus 2% sucrose (STAA) to produce an abundant density of M. oryzae conidia. REFERENCES
Bhattacharyya D, Bose SK, 1981. Studies on standardizing the condition of sporulation in Pyricularia oryzae. Indian Phytopathology, 34: 382-383 Couch BC, Kohn LM, 2002. A multilocus gene genalogy concordant with host preference indicates segregation of a new species, Magnaporthe oryzae from M. grisea. Mycolgia 94: 683-693 Dhingra OD, Sinclair JB, 1995. Basic Plant Pathology Methods.2nd ed. Boca Raton FL. CRC Press. Latterell, FM, Rossie AE, 1986. Longevity and pathogenic stability of Pyricularia oryzae. Phytopathology 76: 231-235 Ou SH, 1980. Pathogen variability and host resistance in rice blast disease. Annual Reveiw.Phytopathology. 18:167-187 Romero A, Carrion G, Rioco-Gray V, 2001. Fungal latent pathogens and endophytes from leaves of Partheium hysterophorus (Asteraceae). Fungal Diversity 7: 81-87 SAS Institute 2007, SAS/STAT Users Guide. Release 9.1.Cary, NC, USA: SAS Institute. Satyanarayana K, Reddy CS, 1985. A new cheap medium supporting the sporulation of Pyricularia oryzae CAV. Indian Journal of Mycology and Plant Pathology. 16: 329-330 Smiley RW, Dernoden PH, Clarke BB, 1996. Compendium of Turfgrass Diseases. St. Paul, MN. The American Phytopathological Society. Snyder WC, 1947. Advantages of natural media and environment in the culture of fungi. Phytopathology 37: 420-421 Tredway LP, Stevenson KL, Burbee LL, 2003. Components of resistance to Magnaporthe grisea in Coyote and Coronado tall fescue. Plant Disease 87: 906-912 Vincelli P, Dixon E, 2002. Resistance to QoI (stobilurin-like) fungicides in isolates of Pyricularia griesea from perennial ryegrass. Plant Disease 86: 235-240 Yorionori JT, Thurston HD, 1974. Sporulation of pyricularia oryzae on crushed rice leaves. Fitopathologia 9: 24-27

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