D. H. Jiang

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.

Genome characterization of a debilitation-associated mitovirus infecting the phytopathogenic fungus Botrytis cinerea

The full-length sequences of Botrytiscinereamitovirus 1 (BcMV1) and an associated RNA (BcMV1-S) in strain CanBc-1c-78 of Botrytis cinerea were determined. Sequence analysis showed that BcMV1 is 2804 nt long and AU-rich (66.8%). BcMV1 shares 95% nucleotide sequence identity with Ophiostomanovo-ulmimitovirus 3b (OnuMV3b). However, it is 472 nt longer than OnuMV3b. Mitochondrial codon usage revealed that BcMV1 contains one open reading frame encoding RdRp, which is 96% identical to the RdRp of OnuMV3b. These findings confirm that BcMV1 belongs to the genus Mitovirus and is a strain of OnuMV3b. BcMV1-S is 2171 nt long and derived from BcMV1 through a single internal in-frame deletion of 633 nt, suggesting that it is a defective RNA of BcMV1. BcMV1-S was found to suppress the replication of BcMV1 and to be co-transmissible with BcMV1 through hyphal anastomosis. Its presence, however, did not alleviate the BcMV1-associated debilitation phenotypes of B. cinerea.

Botrytis fabiopsis, a new species causing chocolate spot of broad bean in central China

The current study was conducted to identify Botrytis spp. isolated from symptomatic broad bean plants grown in Hubei Province, China. Among 184 Botrytis strains, three distinct species, B. cinerea, B. fabae and a previously undescribed Botrytis sp., were identified based on morphology of colonies, sclerotia and conidia. The novel Botrytis sp. is described herein as a new species, Botrytis fabiopsis sp. nov. At 20 C B. fabiopsis grew on potato dextrose agar (PDA) at 12–13 mm d−1, similar to B. fabae (13 mm d−1), but slower than B. cinerea (17–19 mm d−1). It formed pale gray colonies with short aerial mycelia and produced gray to black sclerotia in concentric rings on PDA. B. fabiopsis produced greater numbers of sclerotia than B. cinerea but fewer than B. fabae. Conidia produced by B. fabiopsis on broad bean leaves are hyaline to pale brown, elliptical to ovoid, wrinkled on the surface and are larger than conidia of B. fabae and B. cinerea. Phylogenetic analysis based on combined DNA sequence data of three nuclear genes (G3PDH, HSP60 and RPB2) showed that B. fabiopsis is closely related to B. galanthina, the causal agent of gray mold disease of Galanthus sp., but distantly related to B. fabae and B. cinerea. Sequence analysis of genes encoding necrosis and ethylene-inducing proteins (NEPs) indicated that B. fabiopsis is distinct from B. galanthina. Inoculation of broad bean leaves with conidia of B. fabiopsis caused typical chocolate spot symptoms with a similar disease severity to that caused by B. fabae but significantly greater than that caused by B. cinerea. This study suggests that B. fabiopsis is a new causal agent for chocolate spot of broad bean.

Morphological and phylogenetic identification of Botrytis sinoviticola, a novel cryptic species causing gray mold disease of table grapes (Vitis vinifera) in China.

Seventy-five isolates of Botrytis collected from table grapes (Vitis vinifera) with gray mold symptoms in China were identified based on morpho-cultural characteristics on potato dextrose agar (20 C) and/or phylogenetic analysis using the sequences of three nuclear genes (G3PDH, HSP60, RPB2). Isolates of different species of Botrytis were compared with fenhexamid sensitivity, Bc-hch gene-RFLP haplotyping and pathogenicity to V. vinifera. The 75 isolates comprise two species, B. cinerea (63 isolates) and an undescribed Botrytis sp. (12 isolates) described here as Botrytis sinoviticola Zhang et al. sp., nov. Both B. sinoviticola (Bs) and B. cinerea (Bc) were found to have 20 C optimum for mycelial growth and 25 C for conidial germination. Sensitivity to fenhexamid was significantly greater (P < 0.05) for Bc (EC50 = 0.04 ± 0.01 μg mL−1) than for Bs (EC50 = 0.08 ± 0.02 μg mL−1). Digestion of the PCR amplicons of the Bc-hch gene with Hha I generated two haplotypes, Group I haplotype for Bs and Group II haplotype for Bc. Bs infected table grapes (leaves, berries) only through wounds, whereas Bc infected both injured and non-injured tissues of table grapes. This study suggests that Bs is a cryptic species sympatric with Bc on table grapes in China.

Degradation of oxalic acid by the mycoparasite Coniothyrium minitans plays an important role in interacting with Sclerotinia sclerotiorum.

Coniothyrium minitans (Cm) is a mycoparasite of the phytopathogenic fungus Sclerotinia sclerotiorum (Ss). Ss produces a virulence factor oxalic acid (OA) which is toxic to plants and also to Cm, and Cm detoxifies OA by degradation. In this study, two oxalate decarboxylase genes, Cmoxdc1 and Cmoxdc2, were cloned from Cm strain Chy-1. OA and low pH induced expression of Cmoxdc1, but not Cmoxdc2. Cmoxdc1 was partially responsible for OA degradation, whereas Cmoxdc2 had no effect on OA degradation. Disruption of Cmoxdc1 in Cm reduced its ability to infect Ss in dual cultures where OA accumulated. Compared with Chy-1, the Cmoxdc1-disrupted mutants had reduced expression levels of two mycoparasitism-related genes chitinase (Cmch1) and β-1,3-glucanase (Cmg1), and had no detectable activity of extracellular proteases in the presence of OA. On the other hand, the cultural filtrates of the Cmoxdc1-disrupted mutants in OA-amended media showed enhanced antifungal activity, possibly because of increased production of antifungal substances under acidic pH condition resulted from reduced Cmoxdc1-mediated OA degradation. This study provides direct genetic evidence of OA degradation regulating mycoparasitism and antibiosis of Cm against Ss, and sheds light on the sophisticated strategies of Cm in interacting with metabolically active mycelia and dormant sclerotia of Ss.

Characterization of some culture factors affecting oxalate degradation by the mycoparasite Coniothyrium minitans

Aims:  To find possible approaches to utilize the mechanism of oxalate degradation by Coniothyrium minitans (Cm) in controlling the plant pathogen Sclerotinia sclerotiorum (Ss).

Water-assisted dissemination of conidia of the mycoparasite Coniothyrium minitans in soil

Abstract A study was conducted to determine water-assisted dissemination of conidia of Coniothyrium minitans (Cm), a mycoparasite of Sclerotinia sclerotiorum (Ss), in four soils (yellow–brown soil, red-clay soil, fluvo-aquic soil and black soil) and one sand. Conidial suspensions (1×107 conidia mL−1) of Cm were applied to sieved (2 mm screen) soil or sand in glass tubes to test vertical dissemination (VD) and in aluminum boxes to test horizontal dissemination (HD) of conidia. Results showed that conidia of Cm could be disseminated with water and spread in soil or sand for 16–20 cm vertically and for 5–10 cm horizontally. The conidial concentration of Cm was logarithmically reduced with the increase in depth of VD or the distance of HD. Dissemination of Cm conidia in sand was better than that in four soils. Potting experiments were done to further understand the potential of water-assisted dissemination of Cm conidia in suppression of Ss carpogenic germination. Results showed that more apothecia were produced by Ss sclerotia located at the soil surface than those at 5 and 10 cm in depth. The minimum Cm concentration for suppression of Ss carpogenic germination was 1000 conidia g−1 soil. Two-season field trials indicated that water-assisted application of Cm was an effective strategy used at the time for transplanting oilseed rape seedlings to suppress Ss carpogenic germination, thereby reducing the primary infection source for sclerotinia diseases of oilseed rape.

First report of Pestalotiopsis microspora causing leaf blight of Reineckea carnea in Central China.

Pink reineckia (Reineckea carnea (Andrews) Kunth) is an evergreen herbaceous perennial plant widely grown as groundcover or for medical purposes in southern China. In 2006 and 2007, severe leaf blight was observed on pink reineckia in Wuhan, China. On newly formed pink reineckia leaves, symptoms were first noted in early May as grayish to dark brown, oval or irregular-shaped lesions, 1.5 to 0.2 × 0.5 to 0.1 cm (n = 50), on the leaf margin or leaf tip. A yellowish halo surrounded each lesion. Lesions enlarged and coalesced and diseased leaves became blighted during the fall and winter. In severely infected plots, most plants became straw-colored and had to be replaced with healthy seedlings. A fungus was isolated from surface-disinfested lesions on potato dextrose agar (PDA) at a frequency of 85.7%. One of 30 isolates, designated C2, was characterized further. The fungus growing on PDA at 20°C for 14 days formed zonate white colonies and black acervular conidiomata. Conidia of the fungus aggregated on acervuli as droplets. Conidia were fusiform and 20.7 to 32.2 × 5.8 to 9.8 μm (n = 50). Each conidium had one hyaline apical cell, one hyaline basal cell, and three dark brown median cells. There were two to four hyaline filamentous appendages 8.1 to 20.4 μm long attached to each apical cell and one hyaline appendage 2.4 to 7.1 μm long attached to each basal cell. The cultural and morphological characteristics of isolate C2 matched the description for Pestalotiopsis microspora (Speg.) Batista & Peres (1,2). The internal transcribed spacer (ITS) region of the ribosomal DNA (ITS1-5.8S-ITS2) was PCR-amplified and sequenced. The ITS sequence (606 bp) for isolate C2 (GenBank Accession No. EU935587) was 100% similar to P. microspora isolates TA-57 (GenBank Accession No. AY924267) and LK32 (GenBank Accession No. DQ001002). Pathogenicity of isolate C2 was tested with the method described by Keith et al. (2). Four detached leaves were wound inoculated or inoculated without wounding with mycelia on agar plugs (4 mm in diameter; three plugs per leaf) or conidial suspensions (107 conidia per ml; 20 μl on each of three sites per leaf). Control leaves were wound inoculated with PDA or sterile water. All inoculated leaves were maintained in a moist enamel tray under fluorescent light for 7 days at 20°C. The test was performed twice. After 4 days of incubation, necrotic leaf lesions resembling symptoms that occurred in the field were observed on the wound-inoculated leaves, whereas the control leaves and C2-inoculated leaves without wounding remained healthy. Therefore, wounding was necessary for symptom development (2). A fungus was reisolated from the C2-induced leaf lesions and the morphology of colonies and conidia were identical to that for isolate C2 of P. microspora. On the basis of the results of isolations, inoculations, and fungal identification, P. microspora was determined to be the causal agent for leaf blight of pink reineckia occurring in Wuhan, China. This fungus previously has been reported as the causal agent of scab disease of Psidium guajava in Hawaii (2), decline of Torreya taxifolia in Florida (3), and leaf blight of Lindera obtusiloba in Korea (1). To our knowledge, this is the first report of the occurrence of P. microspora on R. carnea. References: (1) Y. H. Jeon et al. Plant Pathol. 56:349, 2007. (2) L. M. Keith et al. Plant Dis. 90:16, 2006. (3) M. W. Schwartz et al. Plant Dis. 80:600, 1996.

First report of onion bulb rot caused by Botrytis aclada in China.

During the spring of 2006, onion bulbs with gray mold symptoms on the surface were observed in a few supermarkets in Wuhan, China. Onions mummified as they decayed. Further surveys of five randomly selected batches of onion bulbs in one of the supermarkets indicated that the disease occurred in all batches and the disease incidence ranged from 6 to 50%. Eight diseased onion bulbs were collected arbitrarily and isolations were made using homemade potato dextrose agar (PDA). Single-spore cultures of the isolated Botrytis sp. were established and maintained on PDA plates at 20°C. The 10-day-old PDA cultures of all of these isolates were gray and covered with abundant beige, ovoid- or oblong-shaped conidia, which were budded from terminal ampullae formed on dichotomously branching conidiophores. Conidia from these isolates measured 7.6 to 10.4 μm long and 4.2 to 5.6 μm wide, with an average of 8.4 × 5.0 μm. No sclerotia were produced from any of these PDA cultures after incubation at 20°C for 30 days. Morphological characteristics of colonies and conidia of these isolates were similar to Botrytis aclada according to the description made by Yohalem et al. (3). Inoculation of healthy onion bulbs with one of the eight fungal strains, OnionBc-15, resulted in gray mold symptoms similar to those observed in the supermarkets. Microscopic examinations showed that the size and shape of conidia that formed on the surface of diseased bulbs of onion were identical to the size and shape of conidia of OnionBc-15, indicating that this isolate can cause onion bulb rot. The isolate OnionBc-15 was further characterized by molecular techniques. Genomic DNA was extracted from mycelia of this strain and used as a template for amplification of two previously reported DNA regions, the internal transcribed spacer (ITS) region of the ribosomal RNA genes and the L45-550 sequence (1), which can be used to distinguish B. aclada and two closely related species, B. allii and B. byssoidea (3). Universal primers ITS1 and ITS4 were used to amplify the ITS region (2). A 539-bp DNA sequence was generated, cloned, and sequenced (GenBank Accession No. EU093077). The sequence contained two SphI restriction sites and was 99% identical in nucleotides to that of B. aclada strain PRI006 (GenBank Accession No. AJ716295). It is different from B. allii and B. byssoidea, which have only one SphI restriction site for the ITS1/ITS4-amplified DNA sequence (2). The Botrytis-specific primers, BA2f and BA1r, were used to amplify the L45-550 sequence (2). A 413-bp DNA sequence was generated, cloned, and sequenced. The sequence did not contain any ApoI restriction sites. This is also similar to B. aclada, but different from B. allii and B. byssoidea, which contains one ApoI restriction site in the BA2f/BA1r-amplified DNA sequence (2,3). On the basis of morphological characteristics and the two molecular features, it is concluded that the isolate OnionBc-15 belongs to B. aclada. To our knowledge, this is the first report on the occurrence of B. aclada causing onion bulb rot in China. References: (1) K. Nielsen and D. S. Yohalem. Mycologia 93:264, 2001. (2) K. Nielsen et al. Plant Dis. 86:682, 2002. (3) D. S. Yohalem et al. Mycotaxon 85:175, 2003.