Pyricularia grisea causes blight of buffel grass (Cenchrus ciliaris) in Queensland, Australia

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1 Tropical Grasslands (999) Volume??, Pyricularia grisea causes blight of buffel grass (Cenchrus ciliaris) in Queensland, Australia ROSS F. PERROTT AND SUKUMAR CHAKRABORTY CSIRO Tropical Agriculture, Cooperative Research Centre for Tropical Plant Pathology, University of Queensland, Australia Abstract This is the first report of Pyricularia grisea causing blight of buffel grass (Cenchrus ciliaris) in Australia. Blight caused by Pyricularia grisea is a major problem on pastures of buffel grass cv. American sown in south Texas, USA and northern Mexico. During a survey of buffel grass pastures in the Baralaba and Banana regions of central Queensland in August 998, plants with blast-like lesions were collected. A strain of P. grisea was isolated from these field samples. Following artificial inoculation, cvv. Gayndah, American and Biloela were all susceptible, but there was variation in virulence among the 3 isolates of the fungus examined. More extensive surveys are necessary to establish the extent and severity of this disease and its possible links with the widespread buffel grass dieback known as ill-thrift recently reported in Queensland pastures. Introduction Buffel grass (Cenchrus ciliaris) is an important pasture grass in the semi-arid regions of Australia. Its productivity under drought conditions has made it a popular choice among graziers. Cultivars Gayndah, American and Biloela were planted extensively in Queensland in the early 950s and early 960s, and are still the most widely used despite the availability of several Correspondence: Mr R.F. Perrott, CSIRO, Tropical Agriculture, 306 Carmody Road, St Lucia, Qld 4067, Australia. Ross.Perrott@tag.csiro.au other cultivars. There is an estimated 2.4 M ha under buffel grass in the three major growing regions in Queensland (Cavaye 99) and a further 5 M ha could be suitable as potential buffel grass pasture (Weston et al. 98). In many areas within Queensland, buffel grass is the only sown grass. Since 993, a dieback of buffel grass, often called ill-thrift, has been reported in pockets of central Queensland south-west of Rockhampton, in the Baralaba district (Graham and Conway 998).The condition affects American and Gayndah more than Biloela. Symptoms of illthrift are the thinning of plants with reddishbronze leaves, commonly seen in patches 0 m across. Patches may coalesce and enlarge to 60 m across. The progress of the symptoms has been reported as a reddish colouration, first appearing at leaf tips, later spreading to the whole leaf, then to the stems, after which the whole plant dies. Researchers at the Queensland Department of Primary Industries (QDPI) have so far ruled out soil fertility problems and nematode damage (Graham 998, personal communication). No soil-borne pathogens were detected in soil taken within affected patches (Julie Mackie, Cooperative Research Centre for Tropical Plant Pathology [CRCTPP] 998, personal communication). In 996, buffel blight caused by Magnaporthe grisea (T.T. Herbert) M.E. Barr (anamorph Pyricularia grisea) reached epidemic proportions in south Texas, USA and northern Mexico in pastures sown exclusively with the cultivar Common T Common T-4464 is known as American in Australia. The widespread use of a buffel grass monoculture for M ha between south Texas and Mexico provided an ideal host for the pathogen, allowing it to build up unhindered causing extensive losses (Ocumpaugh and Rodriguez 998). In Queensland, Pyricularia spp. have been found previously in association with buffel grass.

2 2 Ross F. Perrott and Sukumar Chakraborty Herbarium specimens BRIP 2835 and BRIP 6407 (Plant Pathology Herbarium, QDPI) were collected by Bryan Hacker (CSIRO, Tropical Agriculture) in 979 and Gary Kong (QDPI) in 988, respectively. Pyricularia spp. were isolated for identification only, pathogenicity was not confirmed and fungal cultures were not kept. Epidemics of Pyricularia on buffel grass were then unknown. Until now, Pyricularia cultures isolated from other grasses have turned out to be saprophytes (John Alcorn, QDPI 996, personal communication). In this paper we report how we obtained fungal isolates from a field survey and confirmed their pathogenicity to cvv. American, Gayndah and Biloela under controlled conditions. Pyricularia isolates from QDPI are used for comparison in pathogenicity. Materials and methods Survey, isolation and identification of fungi Samples of buffel grass with blast-like lesions (Ou 985) were collected from the edge of ill thrift affected patches from 3 properties between Baralaba and Banana in central Queensland. Fungal isolations were carried out using 2 methods. In the first method, small pieces of affected leaves covering a lesion and the adjoining healthy area were surface-sterilised in % sodium hypochlorite for 5 minutes followed by 2 washes with sterile water, blotted dry on sterile filter paper, then placed on Potato Dextrose Agar (PDA) (30g/L dehydrated commercial PDA, Sigma Chemical, USA with 5g/L technical agar). In the second method, a leaf fragment with a single lesion was placed on moist filter paper in a petri dish, sealed with parafilm and incubated at 25 C for 20 hours. Conidia projecting from the lesion were picked up using a sterile loop and suspended in a drop of sterile water. The conidial suspension was streaked on water agar (20 g technical agar in L distilled water), and incubated at room temperature for approximately 20 hours. Single germinated conidia were transferred on to fresh PDA. Cultures were identified by morphological characters examined under a light microscope. A culture was also sent to the International Mycological Institute (IMI) to confirm its identity. Inoculation and blight severity Three buffel cultivars, American (Common T- 4464; G. E. Pogue Seed Co., Inc., Kenedy, TX), Gayndah and Biloela, were grown from seed in 7.5 cm pots (4 9 plants per pot) containing a sandy loam soil. Plants were grown in a controlled environment facility at CSIRO Tropical Agriculture, Brisbane, Australia, with 4 h photoperiod, 30 C and 75% Relative Humidity during day and 25 C and 95% Relative Humidity during night time. For the initial pathogenicity assay, 5 single conidial isolates from buffel grass were grown on PDA under black light (4h light period) for 2 weeks. Conidia produced in the cultures were suspended in sterile distilled water. In addition, the pathogenicity of 6 isolates of Pyricularia spp. previously obtained from other grasses by the QDPI were tested on the 3 buffel grass cultivars. Plants were spray-inoculated with a spore suspension using pressure pack sprayers (Preval sprayers; Precision Valve Corporation, Yonkers, New York, USA) until run-off occurred. Spore concentrations are listed in Table. Control plants were sprayed with distilled water. The inoculated plants were incubated by placing pots on trays with free water and covering with perspex tents for 48 hours. Seven days after inoculation, plants showing symptoms were rated for lesions per leaf using a 4-point scale; 0 = no symptoms; = a few blight lesions (<0); 2 = many blight lesions (0 20); and 3 = high number of blight lesions (>20). The pathogen was reisolated from lesions, its morphology was compared with previous observations and inoculated on buffel plants to reproduce blight symptoms to confirm Koch s postulates. To assess differences between Pyricularia isolates, further inoculations of the 3 buffel cultivars were carried out with 3 isolates (CS2260, CS2262 and CS2264) using conidia/ml, and incubated as above. Three replicate pots (5 8 plants per pot) of each cultivar were inoculated. Seven days after inoculation, blight severity was estimated as percentage of leaf area affected. An analysis of variance was used to test for significant difference in resistance in the 3 cultivars.

3 Pyricularia grisea causes blight of buffel grass (Cenchrus ciliaris) in Queensland, Australia 3 Table. Severity rating of isolates of Pyricularia spp. on 3 cultivars of buffel assessed 7 days after inoculation with a conidial suspension using a 4-point rating scale where: 0 = no symptoms; = a few blight lesions (<0); 2 = many blight lesions (0 20); and 3 = high number of blight lesions (>20). Isolate Source Inoculum conc. Severity rating on each cultivar American Gayndah Biloela ( 0 4 conidia/ml) CS2260 P. grisea C. ciliaris cv. American CS226 P. grisea C. ciliaris cv. American 3.0 CS2262 P. grisea C. ciliaris cv. American CS2263 P. grisea C. ciliaris cv. American 3.2 CS2264 P. grisea C. ciliaris cv. American BRIP 5748; P. oryzae Oryza rufipogon BRIP 4879 P. penniseti Pennisetum clandestinum BRIP 5855 P. sp. Pennisetum clandestinum BRIP 5636 P. oryzae Oryza sativa.0 0 Not tested 0 BRIP 5666 P. penniseti Pennisetum clandestinum BRIP 585 P. grisea Brachiaria mutica CONTROL Plant Pathology Herbarium, Queensland Department of Primary Industries. Results Survey, isolation and identification of fungi All 3 properties surveyed, growing mainly American buffel grass, showed patches, 0 m across, with clearly defined boundaries, and dead and dying plants within the almost-circular patches. Patches appeared to be distributed randomly within a paddock. Some of the older patches were almost free of buffel grass. On 2 properties where grazing was restricted, symptoms of leaf spot and blight were present on new growth on plants in the vicinity of the patches. Lesions on leaves and stem were round to elliptical with light brown centres and dark red to dark brown borders. Many fungi grew from lesions when leaf segments were plated on PDA or incubated in petri dish moist chambers. Most of these were common saprophytes. Fungi putatively identified as Pyricularia spp. (Barnett and Hunter 987) were selected for further study and 5 pure cultures were established by isolating single spores. Conidia in these strains were hyaline, septate and pyriform and measured between 23.7 and 28.0 µm (mean 25.9 µm) in length and 7.7 to 9.4 µm (mean 8.6 µm) in width (Figure ). Samples of conidia were measured. These measurements are within the range described by Ellis (97) for P. grisea. Dr P.M. Kirk of the International Mycological Institute (IMI) has confirmed the identity of the isolate CS2260 (CS is the authors CSIRO culture collection) as P. grisea. This culture has been placed in the culture collection of QDPI Plant Pathology Herbarium (BRIP 25865) and the IMI dried reference collection (IMI ). Inoculation and blight severity All 3 cultivars inoculated with the 5 isolates from buffel grass showed blight symptoms similar to those observed in the field. Symptoms first appeared as irregular or elliptical water-soaked lesions within 3 4 days of inoculation or as small dark brown to tan coloured lesions ( 2 mm). Within 5 7 days, typical blast-type symptoms, characteristic of Pyricularia spp. (Ou 985), could be seen on leaf and leaf sheath. These lesions were elliptical or eye-shaped with tapering ends formed along the length of a leaf. Lesion shape can be elongated or irregular with a number of lesions coalescing to give a blighted appearance. Lesions usually had a dark-brown outline with a light centre and measured from a few mm to several cm in length (Figure 2). They were often surrounded by a light-green halo due to extensive chlorosis giving the leaf a pale appearance. Leaf tip damage occurred on younger leaves, which had not fully expanded at the time of inoculation. In case of severe infection, older leaves died off completely, turning light brown without any apparent demarcation between lesions. All 5 isolates obtained from the field survey produced blight lesions on all 3 cultivars

4 4 Ross F. Perrott and Sukumar Chakraborty Figure. Light micrograph of Pyricularia grisea conidia of isolate CS2260 (The bar represents 25 µm). (Table ). Isolate CS2260 appeared to be the most aggressive. In contrast, none of the 6 Pyricularia spp. isolates obtained from the QDPI produced symptoms (Table ). Of the 3 selected isolates, CS2260 and CS2264 were significantly more virulent than CS2262. CS2260 and CS2264 caused over 40% leaf area damage to all 3 cultivars. CS2262 caused severe damage (>40%) to American while Gayndah and Biloela suffered only minor damage from this isolate (Table 2). This variation in virulence between the isolates is evident in the significant (P<0.05) isolate cultivar interaction. American was the most susceptible cultivar with 47% of leaf area diseased; Biloela with 37%, and Gayndah with 33% were relatively less susceptible with no significant difference in blight damage between Biloela and Gayndah (Table 2). Table 2. Disease severity, expressed as a percentage of affected leaf area, in 3 cultivars of Cenchrus ciliaris inoculated with 3 isolates of Pyricularia grisea. Isolate Disease severity American Gayndah Biloela (%) CS ac 47 a 50 a CS a b 3 b CS bc 42 a 47 a Values within a column followed by the same letter are not significantly different at P<0.05 (LSD = 5.8). Discussion This is the first report of Pyricularia grisea causing buffel blight in Australia. In common with recent work in southern Texas and northern Mexico (Rodriguez et al. 999), this work establishes P. grisea as a pathogen capable of causing serious damage to buffel grass. The epidemic of buffel blight in the US and Mexico had resulted from inoculum built up over time on a genetically uniform stand of the cultivar American. Although the disease was observed for several years in southern Texas and northern Mexico before reaching epidemic proportions (Rodriguez et al. 999), symptoms were often attributed to drought or other stresses. In this study, typical blight symptoms were more readily observed on young growth in the field. Symptoms on more mature leaves were often not typical of blights caused by fungal pathogens but represented signs of drought or other stresses. This makes it difficult to separate fungal blights from other related symptoms in the field. Compared with the USA and Mexico, there are many more buffel varieties in use in Australia, and this may help reduce the risk of widespread epidemics. The isolates in this study came from one property near Banana and it is imperative that the severity and distribution of P. grisea-induced

5 Pyricularia grisea causes blight of buffel grass (Cenchrus ciliaris) in Queensland, Australia 5 We have not examined the role of weather in the dispersal and infection of this pathogen. However, under experimental conditions, the fungus spreads rapidly between plants to cause blight symptoms within a few days. Healthy plants, placed near infected plants, developed lesions as a result of dissemination of conidia by sprinkler irrigation. Under field conditions, showery weather may cause rapid spread of the pathogen from a focus of infection to produce a blight patch. Two of the three isolates caused over 40% damage to all 3 cultivars. Our results show that isolates vary in their virulence to the 3 cultivars. There is evidence of a high level of genetic diversity in P. grisea infecting buffel grass in southern Texas and northern Mexico. The level of genetic variation ranged from 5% for within a location to 50% between locations and years (Ocumpaugh and Rodriguez 998). We have initiated an exchange of isolates with the north American researchers to determine, firstly, if the Australian isolates are related to the north American isolates and secondly to determine the extent of genetic variation in the Australian pathogen relative to the north American pathogen population. To achieve these aims, a more extensive survey of Queensland buffel growing areas is needed. Other ill-thrift affected sites will need to be surveyed to examine if there is a link between buffel blight and ill-thrift. Acknowledgements We thank Dr Gavin Graham of the Queensland Department of Primary Industries for assistance with the survey and the CRC for Tropical Plant Pathology for facilities. blight be determined from a more extensive field survey. Figure 2. Leaf lesions on buffel grass (cv. American) caused by Pyricularia grisea isolate CS2260 following artificial inoculation. Arrows show typical blast lesions. References BARNETT, H.L. and HUNTER, B.B. (987) Illustrated genera of imperfect fungi. 4th Edn. (Burgess Publishing Company: Minneapolis, USA). CAVAYE, J. (99) The buffel book. (Queensland Dept. Primary Industries: Brisbane, Australia). ELLIS, M.B. (97) Dematiaceous hyphomycetes. (Commonwealth Mycological Institute: Kew, Surrey, England). GRAHAM, T.W.G. and CONWAY, M. (998) Some sick buffel. TGS News & Views, Tropical Grassland Society of Australia, 4, 6. OCUMPAUGH, W.R. and RODRIGUEZ, O. (998) Pasture forage production: integration of improved pasture species into Texas livestock production systems. Proceedings management of grazinglands in Northern Mexico and South Texas. pp OU, S.H. (985) Rice Diseases. 2nd Edn. (C.A.B. International: Slough, UK). RODRIGUEZ, O., GONZALEZ-DOMINGUEZ, J., KRAUSZ, J.P., ODVODY, G.N., WILSON, J.P., HANNA, W.W. and LEVY, M. (999) First report and epidemics of buffel grass blight caused by Pyricularia grisea in south Texas. Plant Disease, p. 398, 83, 4. WESTON, E.J., HARBISON, J., LESLIE, J.K., ROSENTHAL, K.M. and MAYER, R.J. (98) Assessment of the agricultural and pastoral potential of Queensland. Agriculture Branch Technical Report No. 27, Queensland Department of Primary Industries. (Received for publication??????; accepted??????)