H. C. Huang

Hypovirulence and Double-Stranded RNA in Botrytis cinerea.

ABSTRACT Twenty-one strains of Botrytis cinerea isolated from 13 species of plants grown in China were compared for pathogenicity on Brassica napus, mycelial growth on potato dextrose agar, and presence of double-stranded (ds)RNA. The results showed that the strain CanBc-1 was severely debilitated in pathogenicity and mycelial growth, compared with the 20 virulent strains. A dsRNA of approximately 3.0 kb in length was detected in CanBc-1 and 4 hypovirulent single-conidium (SC) isolates of CanBc-1, but was not detected in the 20 virulent strains of B. cinerea and 4 virulent SC isolates of CanBc-1. Results of the horizontal transmission experiment showed that the hypovirulent trait of CanBc-1 was transmissible and the 3.0-kb dsRNA was involved in the transmission of hypovirulence. Analysis of a 920-bp cDNA sequence generated from the 3.0-kb dsRNA of CanBc-1 indicated that the dsRNA element was a mycovirus, designated as B. cinerea debilitation-related virus (BcDRV). Further analyses showed that BcDRV is closely related to Ophiostoma mitovirus 3b infecting O. novo-ulmi, the causal agent of Dutch elm disease. Mitochondria and cytoplasm in hyphal cells of CanBc-1 became degenerated, compared with the virulent isolate CanBc-1c-66 of B cinerea. This is the first report on the occurrence of Mitovirus-associated hypovirulence in B. cinerea.

Control of Postharvest Botrytis Fruit Rot of Strawberry by Volatile Organic Compounds of Candida intermedia

A study was conducted to identify volatile organic compounds or volatiles produced by Candida intermedia strain C410 using gas chromatography-mass spectrometry, and to determine efficacy of the volatiles of C. intermedia in suppression of conidial germination and mycelial growth of Botrytis cinerea and control of Botrytis fruit rot of strawberry. Results showed that, among 49 volatiles (esters, alcohols, alkenes, alkanes, alkynes, organic acids, ketones, and aldehydes) identified from C. intermedia cultures on yeast extract peptone dextrose agar, two compounds, 1,3,5,7-cyclooctatetraene and 3-methyl-1-butanol, were the most abundant. Synthetic chemicals of 1,3,5,7-cyclooctatetraene; 3-methyl-1-butanol; 2-nonanone; pentanoic acid, 4-methyl-, ethyl ester; 3-methyl-1-butanol, acetate; acetic acid, pentyl ester; and hexanoic acid, ethyl ester were highly inhibitory to conidial germination and mycelial growth of B. cinerea. Inhibition of conidial germination and mycelial growth of B. cinerea by volatiles of C. intermedia was also observed. Meanwhile, results showed that incidence and severity of Botrytis fruit rot of strawberry was significantly (P < 0.01) reduced by exposure of the strawberry fruit to the volatiles from C. intermedia cultures or C. intermedia-infested strawberry fruit. These results suggest that the volatiles of C. intermedia C410 are promising biofumigants for control of Botrytis fruit rot of strawberry.

Ultrastructural Study of Mycoparasitism of Gliocladium Roseum on Botrytis Cinerea

Conidia and germ tubes of Botrytis cinerea were highly susceptible to infection by the mycoparasite Gliocladiurn roseurn, when both organisms were grown as a mixed culture in sterile water containing 8-9×10^(4) pollen grains of alfalfa per ml solution. Scanning electron microscopic studies revealed that infection of conidia and germ tubes occurred through direct penetration by hyphal tips of G. roseum without the formation of appressoria. Transmission electron microscopic studies indicated indentation and rupture of the host cell walls at penetration sites. The parasitized conidia and germ tubes of B. cinerea showed signs of cytoplasmic disintegration and the presence of hyphae of G. roseum.

Biological Control of Blossom Blight of Alfalfa Caused by Botrytis cinerea Under Environmentally Controlled and Field Conditions

Fungal and bacterial antagonists were tested for their inhibition of sporulation of Botrytis cinerea on detached alfalfa florets. Clonostachys rosea, Gliocladium catenulatum, and Trichoderma atroviride were evaluated for protecting young blossoms and pods of alfalfa from infection by B. cinerea in vitro. C. rosea was further tested to control pod rot and seed rot caused by B. cinerea under field conditions. The results showed that four of the tested antagonists, C. rosea, G. catenulatum, T. atroviride, and Trichothecium roseum, could inhibit sporulation by B. cinerea on detached alfalfa florets. Both C. rosea and G. catenulatum were effective in suppression of infection of alfalfa pods by B. cinerea when inoculated on fresh petals of alfalfa at the anthesis stage, and their efficacy was greater than that of Trichoderma atroviride. A significant suppression of B. cinerea by C. rosea and G. catenulatum on pods and seed of alfalfa was observed when they were inoculated on senescent petals at the pod-development stage. Results of a field trial indicated that C. rosea applied to upper parts of alfalfa plants significantly suppressed pod rot and seed rot caused by B. cinerea, and significantly increased seed production of alfalfa in each of 3 years. These studies show that C. rosea has potential as a biocontrol agent for control of alfalfa blossom blight caused by B. cinerea.

Infection of alfalfa pollen bySclerotinia sclerotiorum

Blossom blight, caused bySclerotinia sclerotiorum, has become an important disease of alfalfa (Medicago sativa L.) in seed production areas of western Canada. Studies using light microscopy and scanning and transmission electron microscopy revealed that pollen grains of alfalfa are susceptible to infection byS. sclerotiorum. Ascospores ofS. sclerotiorum germinated readily in water with or without pollen grains. Examinations of ascospore—pollen mixtures incubated at room temperature (20–22°C) for 5 days revealed that numerous pollen grains were infected byS. sclerotiorum by direct hyphal penetration through the equatorial germinative pores or through the exine and intine layers of the pollen wall without the formation of infection cushions or appressoria. After penetration, hyphae ramified within the pollen grains, causing plasmolysis of the cytoplasmic membrane and eventual disintegration of the pollen cytoplasm. The study suggests that alfalfa pollen may play a role in the epidemiology of blossom blight in alfalfa.

First Report of Pink Seed of Common Bean Caused by Erwinia rhapontici

In 2001, a new disease of common bean (Phaseolus vulgaris L.) caused by Erwinia rhapontici (Millard) Burkh. was detected in seed samples from southern Alberta, Canada. Infected seeds had pink or pinkish-brown lesions on the seed coat. The disease was found in great northern (cv. US1140), pink (cv. Viva), and pinto (cv. Othello) beans at low (<0.1%) frequencies. Isolation from surface-sterilized pink seeds resulted in bacterial cultures, which produced a water-soluble pink pigment on potato dextrose agar (PDA). Seven isolates were tested for physiological characteristics using conventional tests (1) and API 50CHE test strips (bioMérieux Canada, St. Laurent, Quebec), and tested for cellular fatty acids using the MIDI system (Newark, DE). All isolates were gram-negative, motile, facultative anaerobic rods with mucoid colonies and produced a pink pigment on PDA. They were positive for citrate utilization, catalase, methyl red, and Voges-Proskauer, and negative for arginine dihydrolase, lysine and ornithine decarboxylases, urease, gelatin liquification, indole production, oxidase, and gas production. Fatty acid profiles matched with E. rhapontici (approximately 30% each 16:0 and 16:1 ω7c/15:0 iso 2OH; 12% 18:1 ω7c: 8% each 17:0 cyclo and 14:0 3OH/16:1 iso; 4 to 5% each 12:0 and 14:0). Isolates were positive for acid production from: N-acetyl glucosamine, l-arabinose, amygdalin, arbutin, cellobiose, esculin (hydrolysis), d-fructose, d-fucose, d-galactose, β-gentiobiose, d-glucose, glycerol, i-myo-inositol, lactose, maltose, d-mannitol, d-mannose, melibiose, d-raffinose, l-rhamnose, ribose, salicin, d-sorbitol, sucrose, trehalose, and d-xylose. These results match published results for E. rhapontici (4). For pathogenicity tests, each isolate was inoculated in 30 pods from six bean plants (cv. US1140) as described for pink seed of peas (2). Each pod was inoculated with 0.1 ml of bacterial suspension, approximately 109 CFU/ml, by injection through the mid-rib at the basal end. The same number of uninoculated and water-inoculated pods served as controls. Plants were kept in the greenhouse (20 ± 5°C) for 4 weeks, after which isolations were done as described above. In duplicate experiments, all isolates caused lesions on pods extending up to 5 cm from the inoculation point with corresponding discoloration of seeds. The frequency of infected seeds varied among isolates, ranging from 20 to 50%. E. rhapontici was reisolated from seeds with lesions, but not asymptomatic seeds. The study concludes that pink seed of common bean is due to E. rhapontici, a pathogen previously reported on peas in Alberta, Canada (2), and Montana (3). References: (1) D. J. Brenner. Bergeys Manual of Systematic Bacteriology, vol.1, Williams and Wilkens, Baltimore, MD, 1984. (2) H. C. Huang et al. Can. J. Plant Pathol. 12:445, 1990. (3) B. K. Schroeder et al. Plant Dis. 86:188, 2002. (4) L. Verdonck et al. Int. J. Syst. Bacteriol. 37:4, 1987.

Ulocladium atrum as a biological control agent for white mold of bean caused bySclerotinia sclerotiorum

Field studies were conducted near Lethbridge, Alberta, Canada, in 2001, 2004 and 2005 to determine the efficacy of the antagonistic fungusUlocladium atrum for control of white mold of bean caused bySclerotinia sclerotiorum. Results of the 3 years of field trials showed that, compared with the untreated control, foliar application of a spore suspension ofU. atrum (300 ml m−2 of 106 spores ml−1 suspension) significantly reduced incidence and severity of white mold, increased seed yield and reduced contamination of bean seed by sclerotia ofS. sclerotiorum. The level of control of white mold observed in the treatment ofU. atrum was similar to that of the mycoparasitic fungusConiothyrium minitans, but lower than the fungicide treatments of Ronilan (vinclozolin) at the rate of 1200 g ha−1 per application in 2001, or Lance (boscalid) at the rate of 750 g ha−1 per application in 2004 and 2005. The potential for use ofU. atrum as a biological control agent for sclerotinia diseases is discussed.

First Report of the Purple Variant of Curtobacterium flaccumfaciens pv. flaccumfaciens, Causal Agent of Bacterial Wilt of Bean, in Canada

Bacterial wilt of bean (Phaseolus vulgaris L.) caused by the yellow and orange variants of Curtobacterium flaccumfaciens pv. Flaccumfaciens (Hedges) Collins & Jones was found in western Canada in 2002 (1). A purple variant was found in a pooled sample of discolored cull seeds of great northern bean (cv. US1140) from a crop grown near Bow Island, Alberta, Canada in 2005. Bacterial colonies isolated from purple seed using modified Burkholders agar (MBA) (3) were convex, glistening, and smooth edged with blue pigment diffusing into the medium. Three isolates (V154, V155, and V254) were identified with conventional tests (2), carbohydrate oxidation (GP Microplates, Biolog Inc., Hayward, CA), and cellular fatty acids (CFA) (MIDI, Inc., Newark, DE). All were grampositive, motile, aerobic rods with yellow colonies producing extracellular blue pigment on MBA when grown at 20 ± 2°C. Bacterial isolates grew at 27°C but grew weakly at 37°C. They were positive for catalase and hydrolysis of hippurate and indoxyl acetate and negative for urease, gelatin liquification, and oxidase. CFA profiles were approximately 48% 15:0 anteiso, 40% 17:0 anteiso, 7% 16:0 iso, and 3% 15:0 iso; with 17:1 anteiso A variable but <1%. Many carbohydrates were oxidized in the Biolog microplates with little acid production. The results match C. flaccumfaciens (2) and the MIDI and Biolog databases, as well as the purple variant of C. flaccumfaciens found in Nebraska, the only previous report of this variant (4). The pathogenicity of the three isolates was tested. Seeds of great northern (cv. US1140) and navy (cv. Morden003) beans were soaked in a bacterial suspension (1 × 108 CFU/ml) or distilled water (control) for 1 h, planted in Cornell mix in root trainers, incubated at 28/22°C (16-h day/8-h night) in a growth cabinet for 14 days, and examined for seedling wilt. The test had three replicates per treatment and 20 seeds per replicate in a completely randomized design. All three isolates were pathogenic to both bean cultivars. The wilt incidences were 51, 57, and 56% on US1140 and 64, 76, and 69% on Morden003 for isolates V154, V155, and V254, respectively. The purple variant of C. flaccumfaciens was reisolated from hypocotyls of wilted seedlings but not from healthy controls. The experiment was repeated using the reisolated bacteria and the results were similar to the first experiment, thus fulfilling Kochs postulates. To our knowledge, this is the first report of the purple variant of C. flaccumfaciens pv. flaccumfaciens in Canada. References: (1) T. F. Hsieh et al. Plant Dis. 86:1275, 2002. (2) K. Komagata et al. Page 1313 in: Bergeys Manual of Systematic Bacteriology. Vol. 2. Williams and Wilkens, Baltimore, MD, 1986. (3) G. A. Nelson and G. Semeniuk. Phytopathology 54:330, 1964. (4) M. L. Schuster et al. Can. J. Microbiol. 14:423, 1968.

First report of pink seed of lentil and chickpea caused by Erwinia rhapontici in Canada.

A new disease of lentil (Lens culinaris Medik.) and chickpea (Cicer arietinum L.) caused by Erwinia rhapontici (Millard) Burkh. was found in seed samples from commercial fields in Saskatchewan, Canada in 2002. Infected seeds had a pink or pinkish-brown discoloration of the seed coat. Isolation from surface-sterilized pink seeds resulted in bacterial cultures that produced a water-soluble pink pigment on potato dextrose agar (PDA). Four isolates from different lentil crops, LRC 8265, LRC 8310, LRC 8309, and LRC 8313 and one isolate from a chickpea crop, LRC 8266, were tested as previously described (2). Results of the tests were identical to those for pink bean isolates of E. rhapontici (2) with the following minor exceptions: all were negative for Voges-Proskauer; LRC 8266 was positive for tagatose; LRC 8266, LRC 8309, and LRC 8313 were negative for lactose; and LRC 8266 and LRC 8309 were positive for 5-keto gluconate. For pathogenicity tests, each isolate was inoculated into 30 pods from 6 lentil plants (cv. Laird), 30 pods from 6 desi chickpea plants (cv. Myles), and 30 pods from 6 kabuli chickpea plants (cv. Sanford) by the method described for pink seed of pea (1) and bean (2). Each pod was inoculated with 0.1 ml (0.2 ml for kabuli chickpeas) of bacterial suspension, approximately 108 CFU/ml, by injection through the mid-rib at the basal end. The same number of uninoculated and water-inoculated pods served as controls. Plants were kept in the greenhouse (20 ± 5°C) for 4 weeks, after which isolations of the pathogen were performed as described above. In duplicate experiments, all the isolates caused pink lesions on pods and seeds of lentil, desi chickpea, and kabuli chickpea. The frequency of infected seeds among the five isolates (four lentil and one chickpea) ranged from 50 to 100% on lentil, 73 to 100% on desi chickpea, and 43 to 100% on kabuli chickpea. E. rhapontici was reisolated from seeds with lesions but not from asymptomatic seeds. The study demonstrates that in addition to pea (1) and common bean (2), E. rhapontici is also the causal agent of pink seed of lentil and chickpea. The observation that lentil isolates can infect chickpea and vice versa suggests that host specificity may be lacking in E. rhapontici. To our knowledge, this is the first record of E. rhapontici on lentil and chickpea. References: (1) H. C. Huang et al. Can. J. Plant Pathol. 12:445, 1990. (2) H. C. Huang et al. Plant Dis. 86:921, 2002.

Effect of Verticillium wilt on forage yield of alfalfa in southern Alberta.

The incidence of Verticillim wilt and the forage yield of 12 cultivars of alfalfa (Medicago sativa) were compared in an irrigated field naturally infested with Verticillium albo-atrum near Lethbridge, Alberta. The alfalfa cultivars were seeded in 1986 and examined annually for Verticillium wilt and forage dry matter yield during 1987-1993. The incidence of wilt varied from year to year, and averaged over the years, the resistant cultivars Barrier, AC Blue J, Pioneer 5444, and Vertus had a significantly (P<0.01) lower incidence than the moderately resistant cultivar Maris Kabul, Admiral, and Trumpetor and the susceptible cultivars Excalibur, WL316, Apollo II, Beaver, and Pacer