Response of different fodder legume species to Colletotrichum trifolii

Colletotrichum trifolii, the causal agent of southern anthracnose, is a major fungal disease in red clover (Trifolium pratense) and lucerne (alfalfa, Medicago sativa), with varietal differences reported. However, results on the reactions of other fodder legumes grown in Central Europe are limited. In this study, a greenhouse test for resistance evaluated the response of T. repens, T. alexandrinum, T. incarnatum, T. resupinatum, Lotus corniculatus, M. lupulina, Onobrychis viciifolia, Vicia sativa, and V. villosa against C. trifolii compared with M. sativa. Conditions of the test were not suitable for T. alexandrinum and the two Vicia species; however, T. repens, T. resupinatum, and L. corniculatus proved highly resistant against the fungus. The other species showed mean plant survival rates of 61.5–84.7%.

First Report of Anthracnose of Malva sylvestris Caused by Colletotrichum trifolii in China

Common mallow (Malva sylvestris L.) is a perennial medicinal plant in the Malvaceae family, which is native to Asia, Europe, and North Africa. In July 2012, typical symptoms of anthracnose disease, with a disease incidence of ~70%, were observed on common mallow in the Medicinal Herb Garden of Shenyang Pharmaceutical University, Liaoning, China. The fungus mainly infected the stalks and leaves of M. sylvestris. Pinpoint, brownish lesions initially appeared at the flowering stage and the disease spread within the field. The lesions on stems gradually enlarged and became dark brown, elliptical, and slightly concave. Concurrently, acervuli and mucilaginous conidial masses of the pathogen appeared on lesions under moist conditions. Conidia were hyaline, one-celled, cylindrical with both ends rounded, and measured 10.0 to 12.5 × 2.5 to 4.0 μm (mean 11.3 × 3.3 μm). The fungus was isolated from symptomatic tissues. Small pieces from leaves and stems were surface disinfested with 70% ethanol and 1.5% sodium hypochlorite for 1 min, then rinsed three times with sterile distilled water, and cultured on potato dextrose agar (PDA) at 25°C. The colonies on PDA had initially white aerial mycelia, and later became greenish black with regularly whorled rings. To confirm Koch's postulates, five 3-month-old plants of M. sylvestris were inoculated with a conidial suspension (105 conidia/ml) prepared from PDA cultures incubated for 14 days. Five non-inoculated plants served as controls. The plants were maintained in the greenhouse at 22 to 25°C and about 75% relative humidity under natural daylight. Typical symptoms on inoculated plants were reproduced after ~10 to 14 days, whereas control plants remained asymptomatic. The pathogen was successfully recovered from symptomatic tissues and re-identified, completing Koch's postulates. The internal transcribed spacer (ITS) and large subunit -28S (LSU) region of rDNA was amplified with primers ITS1/ITS4 and NL1/NL4, respectively, and sequenced. Phylogenetic trees (ITS and LSU) that were obtained using MEGE3.1 with the neighbor-joining method showed that both of the isolates fall in the Colletotrichum trifolii clade. The representative sequences (ITS and LSU) were deposited in GenBank (Accession Nos. KJ155692 and KJ920935). The fungus isolated from symptomatic tissues was identified as C. trifolii on the basis of morphological, cultural characteristics, and sequence analysis (2). According to previous references, C. orbiculare and C. malvarum on Malvaceae were respectively described in America and Europe (1,3,4). However, the isolate from M. sylvestris significantly differed from those of C. orbiculare and C. malvarum in cultural characteristics and sequence analysis. In this paper, the results showed that M. sylvestris is a new host of C. trifolii. To our knowledge, this is the first report of mallow anthracnose caused by C. trifolii in China. References: (1) J. A. Bailey et al. Phytopathology 86:1076, 1996. (2) U. Damm et al. Fungal Divers. 61:29, 2013. (3) K. Hyde et al. Fungal Divers. 39:147, 2009. (4) L. Tosi et al. Plant Dis. 88:425, 2004.

First Report of Anthracnose on Alfalfa Caused by Colletotrichum linicola in Serbia

Alfalfa (Medicago sativa L.) is economically the most important forage crop in Serbia. In July 2009, alfalfa plants showed symptoms characteristic of anthracnose disease (“shepherd's crook”) including wilting and death of the upper portion of the stems. Anthracnose of alfalfa has been reported to be caused by Colletotrichum trifolii or C. destructivum (2). Alfalfa plants with anthracnose symptoms were collected in Srpska Crnja, South Banat District, Serbia. Infected tissue samples were surface disinfected with 5% sodium hypochlorite for 2 min and washed three times for 5 min in sterile distilled water. Surface sterilized tissue was transferred to sterile filter paper and placed on potato dextrose agar (PDA), and incubated at 24°C in the dark for 10 days (1). Developing colonies were light to dark olive green. In cultures on PDA medium, acervuli were formed. Conidia from acervuli were released in mucous masses that were orange to cream-pink in color. Conidia were hyaline, aseptate, straight with one end pointed and the other slightly rounded, measuring 12.5 to 25.0 × 2.5 to 7.5 μm (mean 19.83 × 4.42 μm). After 5 days, numerous setae were formed. The setae were slightly darker at the bottom and lighter at the top, septate with 3 septa. Setae dimensions were 100 to 185.5 × 2.5 to 5 μm (average 160.9 × 3.12 μm). The isolated fungus was designated Coll-44. Stems of 30 7-week-old plants were spray-inoculated in the laboratory with an aqueous suspension of conidia (106 spores per ml; 10 ml per plant) harvested from 7-day-old cultures grown on PDA. The plants and two non-inoculated check plants were placed in a greenhouse and a covered with plastic bags at 25°C in darkness. After 48 h, plastic bags were removed from the all plants. All plants were watered once a day. Symptoms were observed 10 days after inoculation. No symptoms were observed on non-inoculated plants. In the greenhouse, all 30 inoculated plants became diseased with anthracnose symptoms after 10 days. Coll-44 was consistently re-isolated from diseased stem tissue. Koch's postulates were fulfilled by re-isolation from inoculated alfalfa plants. Pure culture of the Coll-44 isolate was deposited in the public collection of CBS-KNAW Fungal Biodiversity Centre, Utrecht, the Netherlands (specimen no. CBS 3263). Partial sequences of the internal transcribed spacer regions-ITS (GenBank Accession No. JX908364) and betatubulin-TUB2 gene (KJ556347) were amplified and sequenced from extracted fungal DNA with primer pairs ITS1-ITS4 (4) and T1-Bt2b (3), respectively. ITS sequence of the Coll-44 isolate showed 100% nucleotide identity to the GenBank accessions JQ005765 and AB046609 of C. linicola. TUB2 sequence of isolate Coll-44 showed 99.6% nucleotide identity with the GenBank accession JQ005849 of C. linicola isolate CBS 172.51. To our knowledge, this is the first report of C. linicola causing alfalfa anthracnose in Serbia. References: (1) A. P. Baxter et al. S. Afr. J. Bot. 2:259, 1983. (2) K. D. Hyde et al. Fungal Divers. 39:1, 2009. (3) K. O'Donnell and E. Cigelnik. Mol. Phylogenet. Evol. 7:103, 1997. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

First Report of Race 2 of Colletotrichum trifolii Causing Anthracnose on Alfalfa (Medicago sativa) in Wisconsin

Anthracnose of alfalfa (Medicago sativa), caused by Colletotrichum trifolii, is widespread in the United States. In addition to loss of forage due to death of stems, the pathogen causes crown rot, reducing stand life and winter survival (2), making it one of the most serious diseases of alfalfa. Three physiological races have been described (2). Race 1 is reported to be the dominant race that is present wherever alfalfa is grown, while race 2 was reported in a limited area in the Mid-Atlantic states, and race 4 was found in Ohio (1). Conspicuous, straw-colored dead stems with a “shepherd's crook” wilt and large, sunken, diamond-shaped lesions with a dark border were observed in experimental plots and breeding nurseries of experimental lines in Clinton and West Salem, WI, in August 2011 and in West Salem, WI, in mid-August 2012. Acervuli with black setae and orange spore masses were observed in lesions placed in moist chambers for 2 days at room temperature with ambient room lighting. Conidia were germinated on 1% water agar and then single hyphae were transferred to potato dextrose agar (PDA) plates. DNA was extracted from pure cultures of strains DA-1 (Clinton, WI) and FGI-3 (West Salem, WI), the rDNA ITS1-5.8S-ITS2 region was amplified with primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′), the products sequenced directly, and the sequences compared to the ITS region of known race 1 and race 2 strains of C. trifolii. The sequences from DA-1 and FGI-3 were identical to the ITS sequence of C. trifolii 2sp2 (race 1; KF444778) and C. trifolii SB-2 (race 2; KF444779), but distinct from the ITS sequence of C. destructivum (JQ005764) and C. dematium (JX567507), which can cause anthracnose on alfalfa (1). Conidia from DA-1 and FGI-3 were harvested from 7-day-old cultures grown on PDA plates, diluted to 2 × 106 conidia/ml, and sprayed to runoff on 10-day-old growth chamber grown plants of three differential cultivars: Saranac (susceptible to races 1 and 2), Arc (resistant to race 1, susceptible to race 2), and Saranac AR (resistant to races 1 and 2). Plants were maintained at 100% relative humidity for 48 h and then grown in a growth chamber at 24°C with a 16-h photoperiod. Symptoms were rated at 14 days after inoculation. In the three repetitions of the experiment using 75 plants of each cultivar in each experiment, less than 10% of the Saranac and Arc plants survived, while survival of Saranac AR was 31 to 44%. The approximate expected survival of differential cultivars inoculated with race 1 is 1% for Saranac, 65 to 70% for Arc, and 45% for Saranac AR (2). Aggressiveness of race 2 strains on Saranac AR is variable, ranging from 12 to 68% plant survival (3). The susceptibility of Arc when inoculated with DA-1 and FGI-3 is consistent with the reaction to race 2 strains, indicating that both strains are race 2. The isolation of race 2 strains in major alfalfa growing regions in Wisconsin indicates that this physiological race is currently more widespread than previously observed. Although most modern alfalfa cultivars have resistance to race 1, few cultivars with resistance to race 2 are available. The occurrence of C. trifolii race 2 in the Midwest United States should be considered in alfalfa breeding programs when developing multi-pest resistant alfalfa cultivars. References: (1) J. J. Ariss and L. H. Rhodes. Plant Dis. 91:1362, 2007. (2) N. R. O'Neill. Plant Dis. 80:450, 1996. (3) N. R. O'Neill et al. Phytopathology 79:750, 1989.

Effect of Glyphosate Application on Foliar Diseases in Glyphosate-Tolerant Alfalfa

Glyphosate, the active ingredient in Roundup herbicide, inhibits 5-enol-pyruvyl shikimate 3-phophate synthase (EPSPS), an enzyme found in plants, fungi, and bacteria. Plants engineered for glyphosate tolerance with a glyphosate-insensitive EPSPS take up and translocate the herbicide throughout the plant. In greenhouse experiments, we found that application of glyphosate at the recommended field application rate completely controlled alfalfa rust (Uromyces striatus) on 4-week-old plants inoculated with the fungus 3 days after glyphosate treatment. Control was effective in all seven cultivars tested. The level of protection declined with time after application, indicating that control transitory and protection declined with time after inoculation, suggesting that protective treatments have fungistatic activity. Complete control of rust was obtained when glyphosate was applied up to 10 days after inoculation with rust spores, indicating that the herbicide also has curative activity. Treatment increased protection from anthracnose, caused by Colletotrichum trifolii, a hemibiotrophic pathogen, and reduced symptom severity for spring black stem and leaf spot, caused by Phoma medicaginis, a necrotrophic pathogen. These results indicate that glyphosate could be used to help manage foliar diseases in glyphosate-tolerant alfalfa.

Resistência de cultivares de alfafa à antracnose e à mancha de leptosphaerulina em Uberlândia-MG

Tendo em vista a importância da alfafa como forrageira para a produção de feno de alta qualidade, avaliou-se a susceptibilidade de 27 cultivares às doenças de parte aérea bem como progresso da doença nas condições climáticas da região de Uberlândia-MG. As doenças diagnosticadas foram: antracnose (Colletotrichum trifolii Bain & Essary) e mancha de leptosferulina (Leptosphaerulina briosiana (Pollaci) J. H. Graham & Luttrel). Os resultados indicaram as cultivares BR1, MONARCA, SUTTER, MARICOPA, BR4, SW821O, 5929 e MH15 mais resistentes às lesões de leptosferulina. As cultivares ARAUCANA, IC1990, MH15 E SW8112A mostraram-se resistentes à antracnose. As cultivares Maricopa e ICI990 não apresentaram desfolha. O trabalho apresentou uma correlação simples significativa entre severidade da antracnose e porcentagem de desfolha. O modelo quadrático adequou-se para a curva de progresso da doença da mancha de leptosferulina.

Molecular and morphological determination of Colletotrichum trifolii isolates originating from alfalfa

Colletotrichum trifolii is a fungal pathogen responsible for anthracnose disease in alfalfa. The isolates of C. trifolii were obtained from diseased alfalfa stems collected from field in Serbia. It was determined by pathogenicity examination that four isolates (Luc-7, Luc-17, Luc-27, Luc-33) can cause stem lesions on inoculated alfalfa plant. Our isolates were compared using reference isolates of C. trifolii (CBS 158.83). Isolates on MA and CDA developed olive green to grey colonies with white margin, while the substrate got dark olive green color. Conidiophores were hyaline, varied in length and produced a succession of conidia apically. Conidia were hyaline, straight, rounded at the ends, and non-septated with average size 7.85x3.87 ?m. Average sizes of appressoria were 7.5-16.5 x 5.5 x 8.9 ?m. The polymerase chain reaction (PCR) with one set of specific primers was used for the detection of examined isolates of Colletotrichum trifolii. Amplification of desired DNA fragment (590 bp) was determined using specific primer pair TB3-F/TB3-R. Achieved results of amplification indicated that the isolates Luc-7, Luc-17, Luc-27, as well as CBS 158.83, had traits of C. trifolli. Amplification and band on about 430 bp appeared in isolate Luc-33, which also belonged to C. trifolli species.