绿色木霉菌株Tvir-6对黄瓜根结线虫的防治效果研究

从土壤中分离到木霉菌株Tvir-6,结合形态学和分子生物学的ITS和tef1序列扩增法鉴定该木霉菌株为绿色木霉(Trichoderma viride)。通过发酵液试验、盆栽和田间试验分析了绿色木霉菌株Tvir-6对根结线虫的防治效果,结果表明,绿色木霉菌株Tvir-6发酵液在24 h内对根结线虫二龄幼虫的校正死亡率高达98.5%,在盆栽试验中根结减退率为78.2%,在田间试验中防效达到64.9%,与10%噻唑膦颗粒剂防治效果相近,并可使黄瓜产量提高34.1%。绿色木霉菌株Tvir-6的筛选及作用效果研究丰富了黄瓜根结线虫生防菌资源,具有重要应用潜力,为防治黄瓜根结线虫提供了重要材料及依据。

木霉菌对根结线虫和孢囊线虫防治机理研究进展

根结线虫Meloidogyne spp.和孢囊线虫Heterodera spp.是分布最广、危害最严重的两类植物病原线虫。它们寄生于植物根部,通过巨型细胞或合胞体获取营养,影响植物生长发育,对农作物造成严重的经济损失。木霉菌Trichoderma spp.是农业生产中重要的生防资源。近年来,随着环境保护意识的提升,木霉菌作为植物寄生线虫的生防资源越来越受到重视。本文主要从木霉菌对根结线虫和孢囊线虫的生防机制、作用方式、影响因素及存在的问题等方面进行综述,分析木霉菌在生物防治中存在的问题,并对其应用前景进行展望。

Chinese leek(Allium tuberosum Rottler ex Sprengel) reduced disease symptom caused by root-knot nematode

Root-knot nematodes（RKNs,Meloidogyne spp.） are obligate biotrophic parasites that settle close to the vascular tissues in plant roots.The diseases resulting from RKN infections cause serious damage to agricultural production worldwide.In the present paper,the resistance of Chinese leek（Allium tuberosum Rottler ex Sprengel） against RKNs,its suppressive effect on nematode disease,its nematicidal activity and its component profile were studied to identify an efficient disease control method.In soil heavily infected by nematodes,Chinese leek showed strong resistance to RKNs.Additionally,the gall indexes of cucumber plants rotated with Chinese leek and of tomato plants intercropped with Chinese leek were reduced by 70.2 and 41.1%,respectively.In a pot experiment,the gall indexes of Chinese leek extract-treated tomato and cucumber plants were reduced by 88.9 and 75.9%,respectively.In an in vitro experiment,the mortality rate of a RKN（Meloidogyne incognita J2） treated with Chinese leek extract was significantly higher than that of the control.The gas chromatography-mass spectrometry（GC-MS） analysis revealed that glycosides,carboxylic acids,ketones and organic sulfides are the main components in the Chinese leek extract.This study revealed that Chinese leek possesses a high resistance to RKNs,has strong nematicidal activity against M.incognita and can significantly reduce the incidence of disease caused by nematodes.

A Meloidogyne incognita effector Minc03329 suppresses plant immunity and promotes parasitism

Meloidogyne incognita is a devastating plant-parasitic nematode.Effectors play important roles during the stages of nematodes infection and parasitism,but their molecular functions remain largely unknown.In this study,we characterized a new effector,Minc03329,which contains signal peptide for secretion and a C-type lectin domain.The yeast signal sequence trap experiments indicated that the signal peptide of Minc03329 is functional.In situ hybridization showed that Minc03329 was specifically expressed in the subventral esophageal gland.Real-time qPCR confirmed that the expression level of Minc03329 transcript was significantly increased in pre-parasitic and parasitic second-stage juveniles(pre-J2s and par-J2s).Tobacco rattle virus(TRV)-mediated gene silencing of Minc03329 in host plants largely reduced the pathogenicity of nematodes.On the contrary,ectopic expression of Minc03329 in Arabidopsis thaliana significantly increased plant susceptibility to nematodes.Transient expression of Minc03329 in Nicotiana benthamiana leaves suppressed the programmed cell death triggered by the pro-apoptotic protein BAX.Moreover,the transcriptome analysis of Minc03329-transgenic Arabidopsis and wild type revealed that many defense-related genes were significantly down-regulated.Interestingly,some different expressed genes were involved in the formation of nematode feeding sites.These results revealed that Minc03329 is an important effector for M.incognita,suppressing host defense response and promoting pathogenicity.

Bacterial artificial chromosome library construction of root-knot nematode resistant pepper genotype HDA149 and identification of clones linked to Me3 resistant locus

Pepper （Capsicum annuum. L.） is a widely cultivated vegetable crop worldwide and has the second largest planting area and the first largest vegetable output and value in China. Pepper root-knot nematode （Meloidogyne spp.） is one of the most serious pests of pepper, which caused huge losses every year. Previous studies showed that the Me3 gene is resistant to a wide range of Meloidogyne species, including M. arenaria, M. javanica, and M. incognita. HDA149, a double haploid pepper genotype, harboring the root-knot nematode resistance gene Me3, was used to construct bacterial artificial chro- mosome library （BAC） via the vector of CopyControFM pCC1 in this study. The library consists of 210 200 BAC clones and is equivalent to 5.3 pepper genomes. The average insert size is 95 kb, and most of them are 90-120 kb; but the empty clones are less than 3%. In order to screen the BAC library easily, 550 super pools with 384 BAC clones of each pool were further developed in this study. Specific primers from Me3 gene locus were used for BAC library screening, and more than 20 positive BAC clones were obtained. Then the selected positive BAC clones were analyzed by restriction enzyme digestion, BAC-end sequencing, marker development, and new positive BAC clones exploration, respectively. Finally, the contig with total length of about 300 kb linked to the Me3 locus was constructed based on chromosome walking strategy, which made a solid foundation for the cloning of the important root-knot nematode resistance gene Me3.

Expression of mitogen-activated protein kinase double-stranded RNA in cucumber has no apparent effect on the diversity of rhizosphere archaea

The expression of mitogen-activated protein kinase （mapk） double-stranded RNA in cucumber is effective in controlling infestations of the root-knot nematode Meloidogyne incognita. However, little is known about the ecological effects of transgenic plants. Here, we analyzed the diversity of 16S rDNA genes derived from the rhizosphere archaea of transgenic cucumber plants as an indicator of ecological change. A total of 17 and 18 operational taxonomic units were detected in the rhizospheres of non-transgenic cucumber and mapk dsRNA-expressing plants, respectively. No significant difference was observed between the two groups according to Shannon and Simpson indices. In soil samples of the two rhizospheres, the dominant group was Crenarchaeota at the phylum level, with Staphylothermus, Methanococcus, Pyrodictium and Sulfolobus the abundant taxa at the genus level. These results suggest that expressing mapk double-stranded （ds） RNA in cucumber has no apparent effect on the diversity of rhizosphere archaea, and provide powerful evidence for the ecological safety of transgenic cucumber expressing mapk dsRNA.

Rediscovery and analysis of Phytophthora carbohydrate esterase(CE) genes revealing their evolutionary diversity

A continuous co-evolutionary arms-race between pathogens and their host plants promotes the development of pathogenic factors by microbes, including carbohydrate esterase(CE) genes to overcome the barriers in plant cell walls. Identification of CEs is essential to facilitate their functional and evolutionary investigations; however, current methods may have a limit in detecting some conserved domains, and ignore evolutionary relationships of CEs, as well as do not distinguish CEs from proteases. Here, candidate CEs were annotated using conserved functional domains, and orthologous gene detection and phylogenetic relationships were used to identify new CEs in 16 oomycete genomes, excluding genes with protease domains. In our method, 41 new putative CEs were discovered comparing to current methods, including three CE4, 14 CE5, eight CE12, five CE13, and 11 CE14. We found that significantly more CEs were identified in Phytophthora than in Hyaloperonospora and Pythium, especially CE8, CE12, and CE13 that are putatively involved in pectin degradation. The abundance of these CEs in Phytophthora may be due to a high frequency of multiple-copy genes, supporting by the phylogenetic distribution of CE13 genes, which showed five units of Phytophthora CE13 gene clusters each displaying a species tree like topology, but without any gene from Hyaloperonospora or Pythium species. Additionally, diverse proteins associated with products of CE13 genes were identified in Phytophthora strains. Our analyses provide a highly effective method for CE discovery, complementing current methods, and have the potential to advance our understanding of function and evolution of CEs.

Characterization and function of Tomato yellow leaf curl virus-derived small RNAs generated in tolerant and susceptible tomato varieties

supported by the Specialized Research Fund for the Doctoral Program of Higher Education of China （20134320120013）;the Natural Science Foundation of Hunan Province, China （14JJ3095）