Functional Characterization of Verticillium dahliae Race 3-Specific Gene VdR3e in Virulence and Elicitation of Plant Immune Responses.

Verticillium dahliae is a soilborne fungal pathogen that causes disease on many economically important crops. Based on the resistance or susceptibility of differential cultivars in tomato, isolates of V. dahliae are divided into three races. Avirulence (avr) genes within the genomes of the three races have also been identified. However, the functional role of the avr gene in race 3 isolates of V. dahliae has not been characterized. In this study, bioinformatics analysis showed that VdR3e, a cysteine-rich secreted protein encoded by the gene characterizing race 3 in V. dahliae, was likely obtained by horizontal gene transfer from the fungal genus Bipolaris. We demonstrate that VdR3e causes cell death by triggering multiple defense responses. In addition, VdR3e localized at the periphery of the plant cell and triggered immunity depending on its subcellular localization and the cell membrane receptor BAK1. Furthermore, VdR3e is a virulence factor and shows differential pathogenicity in race 3-resistant and -susceptible hosts. These results suggest that VdR3e is a virulence factor that can also interact with BAK1 as a pathogen-associated molecular pattern (PAMP) to trigger immune responses. IMPORTANCE Based on the gene-for-gene model, research on the function of avirulence genes and resistance genes has had an unparalleled impact on breeding for resistance in most crops against individual pathogens. The soilborne fungal pathogen, Verticillium dahliae, is a major pathogen on many economically important crops. Currently, avr genes of the three races in V. dahliae have been identified, but the function of avr gene representing race 3 has not been described. We investigated the characteristics of VdR3e-mediated immunity and demonstrated that VdR3e acts as a PAMP to activate a variety of plant defense responses and induce plant cell death. We also demonstrated that the role of VdR3e in pathogenicity was host dependent. This is the first study to describe the immune and virulence functions of the avr gene from race 3 in V. dahliae, and we provide support for the identification of genes mediating resistance against race 3.

Effects of Temperature and Moisture on the Infection and Development of Apple Fruit Rot Caused by Phytophthora cactorum.

Phytophthora fruit rot, caused by Phytophthora cactorum, is an important disease of apple in China, often causing more than 50% fruit rot in rainy years. We examined the effects of temperature and moisture on the development of the disease and effects of the variables on zoospore release and germination, infection, and lesion development. In vitro, a temperature range of 5 to 20°C had no significant effects on zoospore release dynamics but did significantly affect the quantities of released zoospores. The largest quantity of zoospores was released at 9.9°C according to a fitted model. Zoosporangia released zoospores within 15 min at the test temperatures (0 to 20°C), which peaked at the fourth hour. Zoospores germinated in vitro, requiring free water, at temperatures from 5 to 35°C. The optimum germination temperature was 25.1°C according to a fitted model. The minimum wetness duration required for zoospores to complete the infection process and induce visible lesions on Fuji fruit was 0.40 h at the optimal temperature of 23.0°C according to the fitted model, whereas observed values were 4.5, 1.5, 0.5, 1.5 and 8.5 h at 10, 15, 20, 25, and 30°C, respectively. The number of zoospore infections on fruit at various temperatures and wetness durations were well fitted by the modified Weibull model; based on the model, the optimal temperature for zoospore infections was 23.0°C. Young apple fruit infected by zoospores developed visible lesions from 10 to 30°C, with a predicted optimum of 23.5°C; no lesions developed at 5 or 35°C. The shortest incubation period of the disease was 4 days. These results can be used to develop disease forecasting models for improved fungicide control.

High-Level Production of Exopolysaccharides by a Cosmic Radiation-Induced Mutant M270 of the Maitake Medicinal Mushroom, Grifola frondosa (Agaricomycetes).

A new Grifola frondosa mutant, M270, was successfully isolated for high production of exopolysaccharides (EPSs) using cosmic radiation-induced mutagenesis. We found that the mutant M270 had a clearer and thicker EPS layer (~10 µm) adhering to mycelia than those of its parent strain 265 after Congo red staining. In the 20-L batch fermentation for M270, 10.3 g/L of EPS and 17.9 g/L of dry mycelia biomass were obtained after 204 hours of fermentation. Furthermore, a main water-soluble fraction (EP1) in the EPS was purified from M270 and then confirmed to be heteroglycan-protein complex with 91% (w/w) total carbohydrates and 9% (w/w) total proteins. Four kinds of monosaccharide-D-mannose, D-glucosamine, D-glucose, and D-xylose-were detected in EP1 with a molar ratio of 17.6:1.8:100:2.5. The molecular mass of the main component in EP1 was 8.9 kDa. The EPS from M270 significantly inhibited the growth of sarcoma 180 solid tumors in mice. This G. frondosa M270 mutant could serve as a better candidate strain for polysaccharide production.

Sphingobacterium populi sp. nov., isolated from bark of Populus × euramericana.

A Gram-stain-negative, aerobic, non-motile bacterial strain, 7Y-4T, was isolated from bark tissue of Populus × euramericana. The isolate was able to grow between 10 and 37 °C, with optimal growth occurring at 28-30 °C. Strain 7Y-4T was positive for oxidase and catalase activities, but did not reduce nitrite from nitrate. Positive reactions were observed for the activities of ß-galactosidase, urease and ß-glucosidase, but negative reactions for the activities of gelatinase and the production of indole, acetoin and H2S. Citrate was not utilized. The major fatty acids of strain 7Y-4T are iso-C15 : 0 (28.6 %), C16 : 1ω7c/C16 : 1ω6c (31.8 %) and iso-C17 : 0 3-OH (23.3 %).The major polar lipids of the novel isolate include phosphatidylethanolamine, three unknown phospholipids (PL1-3) and six unknown lipids (L1-6), and the predominant menaquinone is MK-7. The DNA G+C content is 41.7 mol%. Analysis of 16S rRNA gene sequences revealed that the novel isolate shared the greatest sequence similarity with Sphingobacterium hotanense XH4T (93.50 %). On the basis of phenotypic and genotypic characteristics, strain 7Y-4T represents a novel species of the genus Sphingobacterium, for which the name Sphingobacterium populi is proposed. The type strain is 7Y-4T (=CFCC 11742T=KCTC 42247T).

Effects of Temperature, Humidity, and Wound Age on Valsa mali Infection of Apple Shoot Pruning Wounds.

Valsa canker, caused by Valsa mali, is a destructive disease of apple in China. The pathogen infects apple branches, mainly through pruning wounds, and causes branch and tree death. To determine the conditions required for V. mali infection through pruning wounds and growth within the xylem, pruning wounds on 1- to 4-year-old apple branches were inoculated with conidia in vitro under artificially controlled conditions and in vivo in the orchard. The effects of temperature, wetness duration, and wound age on conidial infection through pruning wounds as well as hyphal growth in the xylem were examined. The results showed that, after invading through pruning wounds, V. mali hyphae grew along xylem vessels, tracheids, and rays, expanding longitudinally and laterally. The hyphae could enter adjacent xylem vessels and tracheids through micropores to form a dense hyphal network. Wetness duration did not exhibit an essential effect on conidial infection from pruning wounds. Conidia spread to pruning wounds with rainwater could infect the xylem without any other extra moisture. Temperature for V. mali conidia infection through pruning wounds and hyphal extension in the xylem ranged from 5 to 35°C, with the optimum at 20°C. Pruning wounds made in late March were susceptible to V. mali infection in March, April, and May; the susceptibility was markedly deceased by June, and the pathogen could barely infect through the pruning wounds in November. The infected pruning wounds began to show symptoms from the spring of the following year. More than half of the observed Valsa canker lesions emerged in the spring of the second year, and new canker twigs were also developed from the inoculations in the spring of the third year. March, April, and May are the critical periods for protecting pruning wounds against infection by V. mali in China, and coating pruning wounds with protective film immediately after pruning is an easy and effective measure to protect the pruning wounds.

Brenneria populi sp. nov., isolated from symptomatic bark of Populus×euramericana canker.

Five Gran-stain-negative, facultatively anaerobic, motile, bacterial strains were isolated from symptomatic bark tissue of Populus×euramericana canker. Strains grew at 4-41 °C, pH 4-10 and 0-6 % (w/v) salinity. They were positive with respect to catalase activity and negative for oxidase activity, nitrate reduction and the Voges-Proskauer reaction. Analysis of 16S rRNA gene sequences indicated that these five poplar isolates belong to the genus Brenneria, having highest sequence similarity of 95.98 % with Brenneria goodwinii LMG 26270(T). These five isolates formed a single cluster based on multilocus sequence analysis, indicating that they all belong to a single taxon within the genus Brenneria, which was confirmed by DNA-DNA hybridization. The DNA G+C content was 54.9-55.7 mol%, and the main fatty acids were C16 : 0, C18 : 1ω7c, C17 : 0 cyclo and C16 : 1ω7c/iso-C15 : 0 2-OH. Based on these results, we describe a novel species of the genus Brenneria with the proposed name Brenneria populi sp. nov. The type strain is D9-5(T) ( = CFCC 11963(T) = KCTC 42088(T)).

[Cloning, expression of the CP gene and antiserum preparation of zucchini yellow mosaic virus infecting Benincasa hispida Cogn. var. chieh-qua How].

A Guangzhou isolate of ZYMV infecting Benincasa hispida Cogn. var. chieh-qua How was identified by indicator tests and partial sequence amplification. The coat protein (CP) gene of this virus was amplified by RT-PCR, and ligated to the expression vector pET-22b(+). The recombinant plasmid pET-ZCP was transformed into E. coli BL21 (DE3) and then induced to express by IPTG. It was shown that the CP gene was highly expressed by SDS-PAGE and Western blot analysis. The molecular weight of the recombinant protein was about 33.0 kD. Antiserum with high specificity was produced after the rabbit was immunized with purified recombinant protein, and the titer was determined to be 1/4096 by antigen coating plate-ELISA (ACP-ELISA).