First Report of Magnaporthe poae, Cause of Summer Patch Disease on Annual Bluegrass, in Canada

The ectotrophic, root-infecting fungus Magnaporthe poae Landschoot & Jackson, the causal agent of summer patch disease in the U.S. (2), is implicated in the damage and loss of annual bluegrass (Poa annua L.) on golf course greens. This pathogenic fungus, one of the important root pathogens of turfgrass, attacks and colonizes susceptible turfgrass roots suffering from environmental or cultural stresses. Over 100 turf samples that exhibited symptoms (chlorotic circular or irregular patches of ≥15 cm in diameter with necrotic crowns and discolored roots) reminiscent of summer patch were collected from 77 southwestern Ontario golf courses from July to August of 2009 and 2010. Roots and crowns were often covered with dark, ectotrophic runner hyphae, lobed hyphopodia, and growth cessation structures, characteristic of M. poae. Sections of root tissue were surface sterilized in 0.6% sodium hypochlorite (NaOCl) for 5 min. Sterilized root tissue was plated on potato dextrose agar (PDA) containing 50 mg L–1 streptomycin sulfate and incubated at 28°C for 7 to 10 days. A fungus with morphological characteristics (hyaline mycelium that appears gray or olive-brown when mature) similar to those of M. poae (1) was consistently isolated (≥100 isolates were obtained) and used to identify M. poae through molecular techniques and Koch's postulates. DNA was extracted from the fungal mycelium of the collected isolates using the PowerPlant DNA isolation kit (MO BIO Laboratories, Inc., Carlsbad, CA). The rDNA internal transcribed spacer (ITS) regions of the isolates (≥100 isolates) were amplified by PCR using universal fungal rDNA primers ITS 4 (5′-TCCTCCGCTTATTGATATGC-3′) and ITS 5 (5′- GGAAGTAAAAGTCGTAACAAGG-3′) (3). The purified PCR products were sequenced (GenBank Accession No. JX134588 through JX134601) and a BLAST search exhibited seven isolates with 99% (MAG3, MAG6, MAG13, MAG16, and MAG17) and 100% (MAG1 and MAG14) similarity to M. poae in the GenBank database. Pathogenicity of four isolates (MAG1, MAG3, MAG6, and MAG14) was confirmed with Koch's postulates. Sixteen healthy P. annua core samples (four replicates of each treatment/isolate) collected from an Ontario golf course were inoculated with 25 mg M. poae-infested Kentucky bluegrass seed (Poa pratensis L.; 12.5 mg inoculum applied at the surface of the potting medium and 12.5 mg inoculum applied on the foliar surface) and were placed in a growth chamber with 12-h day/night cycles at 30/25°C and approximate relative humidity. After 2 to 3 weeks, inoculated plants exhibited chlorotic foliage and necrotic roots covered with dark ectotrophic runner hyphae and lobed hyphopodia. Infected root sections from each replication were surface sterilized and placed on PDA containing 50 mg L–1 streptomycin sulfate. The fungal cultures exhibited morphological characteristics consistent with M. poae (1). To our knowledge, this is the first report of summer patch caused by M. poae in Canada. References: (1) B. B. Clarke and A. B. Gould, eds. Turfgrass Patch Diseases Caused by Ectotrophic Root-Infecting Fungi. The American Phytopathological Society, St. Paul, MN, 1993. (2) P. J. Landschoot and N. Jackson. Mycol. Res. 93:59, 1989. (3) T. J. White et al. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Pages 315-322 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al. eds. Academic Press, San Diego, CA, 1990.

First Report of Summer Patch of Kentucky Bluegrass Caused by Magnaporthe poae in Colorado

The ectotrophic, root-infecting fungus Magnaporthe poae is the cause of summer patch of Kentucky bluegrass (Poa pratensis). The disease is widely distributed in the mid-Atlantic Region of the United States and west to central Nebraska and Kansas (2). It also has been found in certain locations of Washington and California (2) but has not been confirmed in the Rocky Mountain Region. In August 2005 and 2006, tan patches and rings of dead turf ranging from 10 to 30 cm in diameter were observed in Kentucky bluegrass swards in Grand Junction and Greeley, CO, respectively. The sites, separated by approximately 360 km, are located west and east of the Continental Divide. A network of ectotrophic hyphae were observed on diseased root segments collected from both sites. A fungus morphologically similar to M. poae (2) was consistently isolated from these segments. DNA was extracted from mycelium of one isolate from each location and amplified by PCR with the M. poae species-specific primers MP1 and MP2 (1). A 453-bp DNA fragment was consistently amplified from DNA of both isolates, diagnostic of M. poae. To our knowledge, this is the first report of summer patch in Colorado and indicates that M. poae may be widely distributed in the central Rocky Mountain Region. References: (1) T. E. Bunting et al. Phytopathology 86:398, 1996. (2) B. B. Clarke and A. B. Gould, eds. Turfgrass Patch Diseases Caused by Ectotrophic Root-Infecting Fungi. The American Phytopathological Society, St. Paul, MN, 1993.

Genetic Relationships Among Magnaporthe poae Isolates from Turfgrass Hosts and Relative Susceptibility of ‘Penncross’ and ‘Penn A-4’ Creeping Bentgrass

Isolates of Magnaporthe poae from turfgrass hosts were analyzed for mating type, genetic relatedness according to ITS sequences, reaction to a previously developed species-specific poly-merase chain reaction (PCR) assay, and virulence on two creeping bentgrass cultivars in growth chamber experiments. Analysis of internal transcribed spacer (ITS) sequences revealed three clades, designated A, B, and C. Clade A contained isolates of both mating types from creeping bentgrass, annual bluegrass, and Kentucky bluegrass. Clade B contained only mating type ‘A’ isolates from annual bluegrass, whereas Clade C contained only mating type ‘a’ isolates from creeping bentgrass. The M. poae PCR assay failed to positively identify several North Carolina isolates from annual bluegrass and creeping bentgrass. M. poae isolates from Kentucky blue-grass were most virulent toward creeping bentgrass in growth chamber experiments. Although isolates of M. poae are not host specific, certain ITS clades may have a limited host or geographical range. The improved creeping bentgrass cv. Penn A-4 was more susceptible to summer patch than cv. Penncross. Additional research is needed to develop methods for accurate diagnosis of summer patch and other patch diseases in creeping bentgrass and to determine how creeping bentgrass cultivars vary in their susceptibility to these root pathogens.

The role of clp-regulated factors in antagonism against Magnaporthe poae and biological control of summer patch disease of Kentucky bluegrass by Lysobacter enzymogenes C3

A global regulator was previously identified in Lysobacter enzymogenes C3, which when mutated, resulted in strains that were greatly reduced in the expression of traits associated with fungal antagonism and devoid of biocontrol activity towards bipolaris leaf-spot of tall fescue and pythium damping-off of sugarbeet. A clp gene homologue belonging to the crp gene family was found to globally regulate enzyme production, antimicrobial activity, and biological control activity expressed by Lysobacter enzymogenes C3 (Kobayashi et al. 2005). Here, we report on the expansion of the biocontrol range of L. enzymogenes C3 to summer patch disease caused by Magnaporthe poae. The clp– mutant strain 5E4 was reduced in its ability to suppress summer patch disease compared with the wild-type strain C3 and was completely devoid of antifungal activity towards M. poae. Furthermore, cell suspensions of 5E4 were incapable of colonizing M. poae mycelium in a manner that was distinct for C3. Strain C3 demonstrated biosurfactant activity in cell suspensions and culture filtrates that was associated with absorption into the mycelium during the colonization process, whereas 5E4 did not. These results describe a novel interaction between bacteria and fungi that intimates a pathogenic relationship.Key words: lytic enzymes, biosurfactant, turfgrass biocontrol agent, mycopathogenic bacteria.

First Report of Summer Patch of Creeping Bentgrass Caused by Magnaporthe poae in North Carolina

An unknown disease was observed in June 2002 and 2003 on creeping bentgrass (CRB [Agrostis stolonifera L.]) putting greens at The Country Club of Landfall in Wilmington, NC that were established in 2001 with a 1:1 blend of cvs. A-1 and A-4. Soil pH ranged from 7 to 8 at this location because of poor quality irrigation water. Symptoms appeared in circular patches of 0.3 to 1 m in diameter that exhibited signs of wilt followed by chlorosis and orange foliar dieback. The disease was initially diagnosed as take-all patch caused by Gaeumannomyces graminis (Sacc.) Arx & D. Olivier var. avenae (E.M. Turner) Dennis, based on the observation of necrotic roots and crowns that were colonized with dark, ectotrophic hyphae. However, the historical lack of take-all patch occurrence in this region led to the suspicion that G. graminis var. avenae was not involved. Sections of root and crown tissue were surface disinfested in 0.6% NaOCl for 5 min or 1% AgNO3 for 1 min and 5% NaCl for 30 s. Tissue was plated on SMGGT3 (2) or on potato dextrose agar containing 50 mg L-1 of tetracycline, streptomycin, and chloramphenicol. A fungus resembling Magnaporthe poae Landschoot & Jackson was consistently obtained regardless of isolation method. Teleomorph production was conducted on Sachs agar (4) overlaid with autoclaved wheat (Triticum aestivum L.) stem sections. Seven isolates were plated alone or paired with M. poae tester isolates 73-1 or 73-15 (3) and incubated at room temperature under continuous fluorescent illumination. Six isolates produced perithecia and ascospores typical of M. poae (3) when paired with 73-15 but not when plated alone or paired with 73-1; these isolates are, therefore, M. poae mating type ‘a’. Isolate TAP42 did not produce perithecia and remains unidentified. Cone-Tainers (3.8 × 20 cm) containing calcined clay were seeded with ‘A-4’ CRB (9.7 g cm-2) and inoculated 8 weeks later by placing four M. poae-infested rye (Secale cereale L.) grains below the soil surface. Inoculated Cone-Tainers were placed in growth chambers with 12-h day/night cycles at 30/25°C, 35/25°C, or 40/25°C. Field plots (1 m2) of ‘A-4’ CRB in Jackson Springs, NC were inoculated on 19 June 2003 by removing a soil core (1.9 × 10.3 cm) from the center of each plot, adding 25 cm3 of M. poae-infested rye grains, and then capping the hole with sand. Growth chamber and field inoculations were arranged in a randomized complete block with four replications. Eight weeks after inoculation in the growth chamber, isolates TAP35, TAP41, and SCR4 caused significant foliar chlorosis and dieback at 12-h day/night cycles of 30/25°C and 35/25°C, but only TAP41 induced symptoms at 40/25°C. Isolate TAP42 did not induce symptoms at any temperature regimen. Orange patches (10 to 15 cm in diameter) were observed in field plots inoculated with TAP41 on 27 August 2003. No other isolates induced aboveground symptoms. Roots and crowns of plants exhibiting foliar symptoms in the greenhouse and field were necrotic and colonized with ectotrophic hyphae, and M. poae was consistently isolated from this tissue. Although M. poae has been associated with CRB in Florida (1), to our knowledge, this is the first report of summer patch of CRB within the normal zone of adaptation for this turfgrass species. Observation of this disease highlights the need for accurate methods for diagnosis of diseases caused by ectotrophic root-infecting fungi. References: (1) M. L. Elliott. Plant Dis. 77:429, 1993. (2) M. E. Juhnke et al. Plant Dis. 68:233, 1984. (3) P. J. Landschoot and N. Jackson. Mycol. Res. 93:59, 1989. (4) E. S. Lutrell. Phytopathology 48:281, 1958.

Impact of Temperature, Osmotic Potential, and Osmoregulant on the Growth of Three Ectotrophic Root-Infecting Fungi of Kentucky Bluegrass

Two isolates each of Magnaporthe poae, Gaeumannomyces incrustans, and Leptosphaeria korrae were grown at 25°C in liquid shake culture in minimal salts medium (-0.12 MPa) or minimal salts medium adjusted to -0.5 MPa with KCl, MgCl2, or polyethylene glycol (PEG). Fungal dry weight of all three species was greater in minimal salts medium amended to -0.5 MPa with MgCl2 than in nonamended medium, and dry weight in medium amended with PEG was not different from dry weights in nonamended medium or medium amended with KCl. Fungi were incubated at varying temperatures on a minimal salts solid-agar medium (-0.12 MPa) adjusted to osmotic potentials ranging from -0.5 to -5.0 MPa with KCl or MgCl2. Optimum growth of M. poae, G. incrustans, and L. korrae on nonamended medium occurred at 30, 30, and 25°C, respectively. At optimum temperatures for each species, fungal growth was greatest at the higher osmotic potentials tested (-0.5 to -1.0 MPa) and decreased in a linear manner as osmotic potential decreased. In most cases, growth was detected at the lowest osmotic potential measured (-5.0 MPa). The relationship of fungal growth to osmotic potential depended on both temperature and osmoregulant. At temperatures optimal or nearly optimal for fungal development, the growth of all three fungi declined more rapidly with decreasing osmotic potential when grown on medium amended with MgCl2 than on medium amended with KCl. At the highest temperature evaluated for growth of M. poae and L. korrae (35 and 30°C, respectively), growth on medium amended with KCl was curvilinear and peaked at osmotic potentials of -2.5 to -3.0 MPa. Furthermore, between osmotic potentials of -2.0 and -5.0 MPa, M. poae grew best at 35°C. When maintained on nonamended minimal salts medium (-0.12 MPa) in liquid culture at 25°C or on nonamended solid-agar medium at temperatures optimal for growth, M. poae grew at a faster daily rate than L. korrae.