Some fungal endophytes from vegetable crops and their anti-oomycete activities against tomato late blight

AIMS: To isolate endophytic fungi from vegetable plants and examine their in vivo anti-oomycete activity against Phytophthora infestans in tomato plants. METHODS AND RESULTS: Endophytic fungi were isolated from surface-sterilized plant tissues and anti-oomycete activity was measured by in vivo assay using tomato seedlings. Endophytic fungi showing potent anti-oomycete activity were identified by morphological characteristics and nuclear ribosomal ITS1-5.8S-ITS2 sequence analysis. A total of 152 isolates were obtained from 66 healthy tissue samples of cucumber, red pepper, tomato, pumpkin and Chinese cabbage and the fermentation broths of 23 isolates showed potent in vivo anti-oomycete activity against tomato late blight with control values over 90%. The Fusarium oxysporum strain EF119, which was isolated from roots of red pepper, showed the most potent disease control efficacy against tomato late blight. In dual-culture tests, it inhibited the growth of Pythium ultimum, P. infestans and Phytophthora capsici. CONCLUSIONS: Among endophytic fungi isolated from healthy tissues of vegetable plants, F. oxysporum EF119 showed the most potent in vivo anti-oomycete activity against tomato late blight and in vitro anti-oomycete activity against several oomycete pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: Endophytic fungi showing anti-oomycete activity in vitro and in vivo may be used as biocontrol agents particularly of tomato late blight.     

马铃薯抗病基因工程研究

马铃薯是世界上最主要的粮食作物之一,但真菌性病害、病毒性病害和细菌性病害等对马铃薯的危害非常严重,成为制约其产量的主要影响因素之一。近年来,随着基因工程的迅速发展,通过转基因的方法在提高马铃薯抗病性方面取得了较大进展,对其进展进行了综述。     

Microbat responses to forest decline

Tree declines have been recorded across forests and woodlands on most continents, causing tree mortality over thousands of square kilometres, yet the impact of tree declines upon mammals have only rarely been quantified. Once the dominant tree over the western parts of the Swan Coastal Plain in Western Australia, tuart (Eucalyptus gomphocephala) forest has been reduced to less than a third of its former range through clearing for agriculture and urban development. Additionally, over the last 30 years, the remnant population has been heavily impacted by a decline that has an unknown cause, but is likely related to a root pathogen coupled with abiotic factors (reduced rainfall, increased salinity and elevated temperatures). Tuart decline is evident as marked canopy dieback, replacement with epicormic growth and increasing bare branches, while leaf litter is lost from the tree surrounds. We examined the effect of tuart decline and other vegetation measures upon bat activity using auditory monitoring. Vegetation structure was correlated with Vespertilionidae bat activity. Falsistrellus mackenziei were more likely to forage around healthy canopies (activity positively correlated with tuart crown density and negatively correlated with tuart crown dieback). By contrast, the other three taxa were more often encountered in declining rather than healthy tuart sites. Chalinolobus gouldii was positively associated with tuart crown dieback. Activity of Vespadelus regulus and Nyctophilus spp. (species not distinguishable from their calls) were significantly positively correlated with an open tall canopy (positively with cover of plants >10 m tall and negatively with overall canopy cover density). There were no vegetation measurements that were strong predictors of activity of two Molossidae species (Ozimops kitcheneri and Austronomus australis), which intercept insects above the forest canopy. This study clearly reveals different factors influencing the activity of bat taxa, which are likely related to where they feed and their manoeuvrability around tree canopies.     

Characterisation of antagonistic Bacillus and Pseudomonas strains for biocontrol potential and suppression of damping‐off and root rot diseases

Novel strains of rhizobacteria, Pseudomonas fluorescens Pf 9A‐14, Pseudomonas sp. Psp. 8D‐45 and Bacillus subtilis Bs 8B‐1, showed broad‐spectrum antagonistic activity and provided suppression of Pythium damping‐off and root rot of cucumber. Their biocontrol potential was further investigated for suppression of additional seedling diseases of cucumber (Phytophthora capsici) and radish (Rhizoctonia solani). Bacterial strains were also characterised for production of antibiotics, metabolites, volatiles, phytohormones and lytic enzymes. Seed and pre‐plant applications of all three antagonistic bacteria as cell suspension and talc or irradiated peat formulations to the infested potting mix provided overall high level of suppression of Phytophthora damping‐off and root rot of cucumber (66–85% healthy seedlings) and relatively low level of suppression of Rhizoctonia damping‐off of radish (18–38% healthy seedlings). Bacterial treatments also resulted in higher plant fresh masses. Seed coating with irradiated peat formulation of a mixture of three bacteria resulted in superior control of Phytophthora damping‐off and root rot of cucumber and much higher plant fresh masses. The presence of genes for biosynthesis of phenazine‐1‐carboxylic acid, 2,4‐diacetylphloroglucinol, pyrrolnitrin and pyoluteorin was confirmed in Pseudomonas strains, and that of fengycin, bacillomycin, bacilysin, surfactin and iturin A in B. subtilis Bs 8B‐1. All three strains produced HCN, salicylic acid, indole‐3‐acetic acid, protease and β‐1,3‐glucanase. Both Pseudomonas strains produced siderophores and only P. fluorescens Pf 9A‐14 showed phosphate solubilisation and chitinase activity. All three strains inhibited pathogen growth by producing volatiles, and gas chromatography–mass spectrometry analysis revealed eight compounds in Pf 9A‐14, 10 in Bs 8B‐1 and 4 in Psp 8D‐45, some with known antifungal activity. The antagonistic and plant‐growth promotion activities of these strains might be due to production of antibiotics, metabolites, lytic enzymes or phytohormones.     

Effect of oxathiapiprolin on asexual life stages of Phytophthora capsici and disease development on vegetables

Phytophthora blight caused by Phytophthora capsici is a serious disease in the production of peppers and other vegetables worldwide. Application of fungicides is an important component in developing effective disease management programmes. However, resistance in P. capsici populations to some commonly used fungicides has been documented. Identification of effective new fungicides with different mode of actions is highly desirable. This study was conducted to determine baseline sensitivity of P. capsici isolates to oxathiapiprolin, the first member of a new class of isoxazoline fungicides, and efficacy of this compound for reduction of Phytophthora blight on bell pepper. A collection of 126 P. capsici isolates were evaluated and all the isolates were sensitive to oxathiapiprolin. EC₅₀ values of oxathiapiprolin in inhibiting mycelial growth, sporangium formation and zoospore germination of 25 selected isolates averaged 0.001, 0.0003 and 0.54 µg mL^?1, respectively. It appeared that asexual life stages of P. capsici were more sensitive to oxathiapiprolin than other compounds used for control of oomycete pathogens. In field studies, oxathiapiprolin applied at different rates through drip irrigation tubes, or by soil drench plus foliar sprays, reduced Phytophthora blight and increased pepper yield significantly. This is the first report of the efficacy of oxathiapiprolin in suppression of P. capsici, which indicates that oxathiapiprolin is effective in inhibiting the pathogen and has the promise to be a viable option for managing Phytophthora blight in bell pepper production.     

Biocontrol of Late Blight Disease (Phytophthora capsici) of Pepper and the Plant Growth Promotion by Paenibacillus ehimensis KWN38

For field application of a bacterial strain used to control Phythophthora capsici, we will need a biologically and economically efficient carrier medium. The known antagonist Paenibacillus ehimensisKWN38 was grown in a grass medium where it showed high antifungal and lytic enzyme activities. To demonstrate the potential of P. ehimensisKWN38 for biocontrol of late blight disease in pepper, pot trials were conducted by treating the 1‐month‐old plants with water (W), a selected grass medium (G3), G plus P. ehimensisKWN38 inoculation (G3P) or synthetic fungicide (F). The shoot dry weight in G3P was higher than that in W and F treatments at 15 days after zoospore infection (DZI). The root dry weight in G3P was also higher than that in W. The root mortality of G3 and W increased over 58 and 80% at 15 DZI, and some plants in those treatments wilted due to the failure of root physiology. The plants in G3P and F survived well because of their better root health conditions. Soil cellulase activity of G3P was consistently higher than that of W and F at earlier observation times (0, 2 and 6 DZI). The root β‐1,3‐glucanase activity of G3P promptly increased to maximum shortly after zoospore infection and reached the maximum value of 51.12 unit g^?1 of fresh weight at 2 DZI. All these results indicate that inoculation of P. ehimensisKWN38 to the root zone of potted pepper plants increases plant growth, root and soil enzyme activities and alleviates the root death caused by infection with P. capsici zoospores.     

福建省马铃薯晚疫病菌线粒体DNA单倍型分析

为明确福建省马铃薯晚疫病菌线粒体DNA单倍型组成和分布情况,采用PCR-RFLP方法分析了2010—2012年从福建省7个地区（福州,长乐,漳州,青口,龙海,霞浦,龙岩）分离的526个马铃薯晚疫病菌株的线粒体DNA单倍型及频率分布。结果表明Ⅰa型为主要单倍型,占72．8％,其次是Ⅱb型,占26．42％,最少的Ⅱa型,占0．76％,没有检测到Ⅰb型。与文献报道历史材料相比较,说明福建省马铃薯晚疫病菌线粒体DNA单倍型的组成发生较大变化,而且福建省马铃薯晚疫病菌线粒体单倍型组成比云南、四川和贵州等地区更为复杂。     

Sequence diversity in the large subunit of RNA polymerase I contributes to Mefenoxam insensitivity in Phytophthora infestans

Phenylamide fungicides have been widely used for the control of oomycete‐incited plant diseases for over 30 years. Insensitivity to this chemical class of fungicide was recorded early in its usage history, but the precise protein(s) conditioning insensitivity has proven difficult to determine. To determine the genetic basis of insensitivity and to inform strategies for the cloning of the gene(s) responsible, genetic crosses were established between Mefenoxam sensitive and intermediate insensitive isolates of Phytophthora infestans, the potato late blight pathogen. F1 progeny showed the expected semi‐dominant phenotypes for Mefenoxam insensitivity and suggested the involvement of multiple loci, complicating the positional cloning of the gene(s) conditioning insensitivity to Mefenoxam. Instead, a candidate gene strategy was used, based on previous observations that the primary effect of phenylamide compounds is to inhibit ribosomal RNA synthesis. The subunits of RNA polymerase I (RNApolI) were sequenced from sensitive and insensitive isolates and F1 progeny. Single nucleotide polymorphisms (SNPs) specific to insensitive field isolates were identified in the gene encoding the large subunit of RNApolI. In a survey of field isolates, SNP T1145A (Y382F) showed an 86% association with Mefenoxam insensitivity. Isolates not showing this association belonged predominantly to one P. infestans genotype. The transfer of the ‘insensitive’ allele of RPA190 to a sensitive isolate yielded transgenic lines that were insensitive to Mefenoxam. These results demonstrate that sequence variation in RPA190 contributes to insensitivity to Mefenoxam in P. infestans.