Viability and pathogenicity of Esteya vermicola in pine trees

Esteya vermicola, as the first reported endoparasitic fungus of pinewood nematode (PWN), exhibited high infectivity in vitro and has been patented based on its potential as a bio-control agent against PWN. The isolation substrates and taxonomic status suggested E. vermicola is associated with beetles, saprotrophic and kills nematodes in trees. However, the direct experimental evidence for this was still lacking. In the present studies, beta-tubulin gene was applied to confirm the taxonomic identification of E. vermicola. Furthermore, our results showed that E. vermicola survived resin and other chemicals secreted by pine trees, and reproduced with new lunate conidia to parasitize other migratory PWNs. In order to confirm the pathogenicity of E. vermicola, pine seedlings and large pine trees were inoculated with 300 µL and 40 mL conidia suspensions (10⁹ mL^?1). The results showed that all treated pine trees were healthy with no differences compared to the controls. Furthermore, necrosis or discoloration caused by this fungus was not observed on wood slices. Basal knowledge was provided for the application of E. vermicola to control PWN in vivo.     

Rapid molecular detection of Esteya vermicola based on specific primers and the FTA-DNA extraction method

Esteya vermicola is an endoparasitic fungus of the pinewood nematode and thus has great biocontrol value. At present, the detection of this fungus is still based on microscopic observations and morphological identification, and the sampling is notably inconvenient and inefficient. In the present study, a pair of specific primers (upstream primer, 5′-GTGCCTCTACCAAGACTCGC-3′; downstream primer, 5′-CGCCAAATGTCAAGATCCGC-3′) was designed to detect E. vermicola. The analysis of the PCR amplification and the agarose gel electrophoresis results led to the establishment of a new method for the detection of E. vermicola through the presence of a 176-bp specific fragment. In addition, the use of a FTA-DNA direct extraction method for the detection of E. vermicola was explored. The results suggest that the proposed method can be effectively used for the rapid detection of E. vermicola and may provide important technical support for follow-up studies of the fungus in field experiments.     

Population dynamics of pinewood nematode and the endoparasitic fungus Esteya vermicola: interactions under experimental conditions

Esteya vermicola (Ophiostomataceae), an endoparasitic fungus, exhibits great potential as a biological control agent against pinewood nematodes (PWNs). The present study reports the interaction between PWNs and E. vermicola at different spore concentrations, number of PWNs and the time of culture. The addition of PWNs enhanced the sporulation of E. vermicola after 10 days of culture. The 5-day-old cultures of E. vermicola prior to addition of PWNs increased the highest amount of sporulation than that of 10- or 15-day-old cultures. The PWNs were completely killed by E. vermicola in the pine tree powder culture medium at the concentrations of 10⁷ and 10⁸ colony-forming-units (CFU) per ml. The interaction of the PWNs and E. vermicola was that PWNs provide nutrition to E. vermicola, however, the PWNs can also feed on mycelium of E. vermicola. The effect of E. vermicola on control of PWNs was determined by the population size, time of pest infection and the duration of co-infection.     

松树萎蔫的成因与控制策略

已报道的松树萎蔫枯死类型有多种,它们之间存在内在的联系,其发生及严重与否涉及多种生态因子,是立地环境、感病松树、媒介昆虫、病原线虫、病原微生物综合作用的结果,基于生态学的可持续有害生物管理应是防治松树萎蔫的有效途径。     

季也蒙假丝酵母对松材线虫病的抑制作用

松材线虫病是重要的森林病害,该病与松材线虫携带的病原菌和松树的内生病原菌密切相关。在室内条件下,初步研究了从人工培养的松材线虫上分离到的菌株C对松材线虫病的抑制作用。在健康的水培马尾松枝上分别接种松材线虫接种液、菌株C接种液、松材线虫与菌株C的混合接种液。处理后松枝的相对重量与相对蒸腾强度均为:接种菌株C的松枝>混合接种的松枝>接种线虫的松枝。处理后15d时,接种线虫的松枝与混合接种的松枝的相对重量有显著性差异(p<0.05)。接种线虫松枝的存活期显著短于其它处理松枝的存活期(p<0.05)。接种菌株C的针叶褐变株数少于接种线虫的松枝,两者有显著性差异(p<0.05)。从接种线虫和混合接种的所有松枝中都分离到松材线虫,且分离出的线虫量没有显著性差异。将8个月生的断根马尾松苗插入菌株C的查彼培养液的滤液中培养,6d后松苗的平均感病指数和感病株率均显著少于对照(p<0.05)。这表明,菌株C对松材线虫病有抑制作用,菌株C培养液中产生的某些代谢物质有利于松苗的抗病和存活。菌株C可能抑制了松树上的内生病原菌和松材线虫携带的病原微生物,或提高了松树的生长力和抗逆能力。经电子显微镜观察并参照AP I 20 C AUX鉴定系统鉴定,菌株C为季也蒙假丝酵母C and id a gu ilierm ond ii。     

林分和景观水平因子对松材线虫病发生的影响——以山东威海地区松林为例

松材线虫病(Pine wilt disease)传入我国以来,造成了大面积松林枯死,严重威胁我国的林业和生态建设的发展,但目前分析林分和景观水平因子对松材线虫病综合影响的研究较少。本文基于松材线虫病危害后期的山东威海地区的松林,利用野外调查数据、森林资源二类调查数据和高分辨率遥感影像,获取了影响松材线虫病危害的林分水平和景观水平的各种因子,通过逐步回归分析、线性回归分析和建立广义线性模型(GLM),探讨了景观分析的最适尺度,分析不同因子在解释松材线虫病危害程度方面的作用和相对重要性,以期为松材线虫病的防控技术及宏观管理政策制定提供科学依据。结果显示,750 m尺度下景观格局指数对松材线虫病发病率的解释效果最好。景观因子中,距疫源地距离、景观分离度与距人为活动区域距离对发病率影响最显著（相对重要性值为1.00）,其中景观分离度和距疫源地距离与发病率呈负相关,距人为活动区域距离与发病率呈正相关;松林斑块连接度的相对重要性次之（相对重要性值为0.69）,与发病率呈正相关;斑块丰富度密度、相关外接圆指数的相对重要性值较低（0.33）。林分因子中,林分密度的相对重要性值最低（0.23）。综合分析林分因子和景观因子的作用发现,在威海地区松材线虫病暴发后期的林分中,相较于林分因子,景观因子起主导作用。研究结果有助于提高对松材线虫病发生的驱动机制的了解。     

Methods for studying the nematophagous fungus Verticillium chlamydosporium in the root environment

In order to exploit fully the biocontrol potential of the nematophagous fungus Verticillium chlamydosporium, it is important to understand the ecology of the fungus in soil, and interactions with both plant and nematode hosts. Several approaches for studying the fungus in soil and the root environment are compared. These include a semi-selective medium for V. chlamydosporium, PCR primers specific for the fungal -tubulin gene, and monoclonal antibodies. In addition to providing a target for species-specific primers, the -tubulin gene is implicated in resistance to the fungicides used in the semi-selective medium, and the genetic basis for this is investigated. Culture and PCR-based methods were used to screen for the presence of the fungus in field soils known to have been suppressive to cereal cyst nematode and that contained V. chlamydosporium populations. Monoclonal antibodies specific for either hyphae or conidia of the fungus were obtained, and their application as a tool for visualising the infection process on the root was explored.     

Role of Bacillus amyloliquefaciens Y1 in the control of Fusarium wilt disease and growth promotion of tomato

The objective of this study was to examine the effects of Bacillus amyloliquefaciens Y1 on the control of Fusarium wilt disease and subsequent improvement in the growth of tomato plants. The Y1 strain strongly inhibited Fusarium oxysporum f. sp. lycopersici in vitro and also produced indole-3-acetic acid (IAA) in both the presence and absence of tryptophan. Over 96% of tomato seeds germinated when treated with either water, tryptone soy broth, or Y1 cultures, whereas root (5.40?cm) and shoot (5.15?cm) lengths were greatest in tomato seedlings treated with Y1 cultures that lacked tryptophan. Three experimental treatments – Black White medium (BW), BW medium with a commercial fungicide (BW?+?F), and Y1 culture inoculated in BW medium (Y1) – were applied to control Fusarium wilt disease under in vivo conditions. Application of Y1 culture and BW?+?F led to significantly lower disease incidence than did BW; moreover, shoot length and fresh and dry weight of both roots and shoots were greater in plants treated with Y1 than in plants treated with either BW or BW?+?F. A similar trend was observed for chitinase and β-1,3-glucanase activities in roots and leaves of tomato plants in all treatment groups over most of the experimental period. Finally, the presence of Y1 in the rhizospheric soils of Y1-treated plants resulted in a significant reduction in the populations of other bacteria. The results of our study demonstrated the effectiveness of Y1 not only in the control of Fusarium wilt disease but also for the enhancement of plant growth in cultivated tomato.     

利用大面积无人机航拍影像研究松材线虫病病死树林间分布规律

本研究使用固定翼无人机拍摄4 200 ha林地,从中选取了广东省河源市和平县阳明镇、紫金县紫城镇、东源县义合镇共3个样地的3 500 ha林地的航拍影像进行分析,用以探究松材线虫Bursaphelenchus xylophilus病死树的空间分布情况,及不同立地因子对疫情的影响,为松材线虫病监测预报提供解决途径。通过Pix4Dmapper软件对航拍的图像进行拼接生成正射影像图(DOM)等成果,然后使用eCognition(易康)软件对影像成果进行分割、分类和信息提取,最后借助ArcGIS平台进行病死树数量统计并获取方位、坡向、坡度、海拔等立地因子信息。结果表明,松材线虫病死树分布均呈聚集分布。使用双对角线法、平行线法、“Z”字法、五点法等不同抽样方法调查发现,仅五点法所得平均数与总体平均数无明显差异(P<0.05)。松材线虫病死树在不同立地因子下均有差异：主要分布在西坡、南坡和东南坡,西坡最多为25.94%,其次是南坡23.57%;主要分布在半阳坡和阳坡,半阳坡占36.54%,阳坡占34.09%;主要分布在凸坡,但随着疫情的发展,凹坡病死树数量逐渐超过凸坡;主要分布海拔区间在30...     

Effects of pine wilt disease invasion on soil properties and Masson pine forest communities in the Three Gorges reservoir region,China

Pine wilt disease (PWD) has caused significant Masson pine mortality in the Three Gorges reservoir region in central China. In this study, five uniform Masson pine stand types infected by PWD were selected and surveyed on slopes and aspects with similar environmental conditions. In sites that had been infected, soil bulk density was reduced, and the difference among the groups was statistically significant (P < 0.05) at the 0–10 cm and 10–20 cm soil layers, but not at 20–40 cm. Other soil water‐related physical properties, excluding noncapillary porosity, significantly differed among the groups in all soil layers. Additionally, the values of available phosphorus, sodium, potassium, calcium, and magnesium were higher in the invaded stands, but the total nitrogen and organic matter contents were lower. Masson pine does not become reestablished following PWD‐induced mortality but is instead replaced by broad‐leaved tree species. Among the 19 examined environmental variables, five were found to be significantly related with the ordination of plant community structure: Masson pine stumps (MPS), K⁺, capillary water holding capacity (CWHC), capillary porosity (CP), and soil water content (SWC). Among these factors, the plant community structure was principally related to MPS and K⁺. The findings of this study show that the outbreak of PWD has impacted Masson pine forest soil properties and altered forest community composition. The disease is negatively related with the presence of Masson pine and positively associated with that of broad‐leaved tree species.     

Fast R-CNN深度学习和无人机遥感相结合在松材线虫病监测中的初步应用研究

松材线虫病因其破坏性强、传播速度快和防治难度大等特点,严重威胁着我国的松林资源.及时发现、定位和清理病死松树是控制松材线虫病蔓延的有效手段.本研究利用小型无人机获得松材线虫病疫点的可见光和多光谱的航摄影像.根据松树针叶颜色变化,将松材线虫Bursaphelenchus xylophilus侵染的松树分为病树和枯死树两种...     

Effect of the combination of bio-organic fertiliser with Bacillus amyloliquefaciens NJN-6 on the control of banana Fusarium wilt disease,crop production and banana rhizosphere culturable microflora

Soil amended with organic amendments has been suggested to be a strategy for managing the Fusarium wilt disease which severely hindered the banana production. The effects of four fertilisation regimes, including chemical fertiliser, manure composts and bio-organic fertiliser (BIO) containing Bacillus amyloliquefaciens NJN-6 for 2-year continuous application on the banana Fusarium wilt disease incidence, crop yield and rhizosphere culturable microbial community were investigated. To explore the soil microflora, plate counting method, in vitro screening method for antagonism, eco-physiological index and culture-dependent denaturing gradient gel electrophoresis method (CD DGGE) were used. The highest banana yield, culturable bacteria, actinobacteria and Bacillus populations, culturable bacteria to fungi (B/F) value, antagonistic Bacillus ratio and lowest Fusarium wilt disease incidence were observed in the BIO treatment. Based on CD DGGE results, the BIO application significantly altered the soil bacteria structure and showed highest richness and diversity. The phylogenetic analysis of the selected bands showed that the most abundant phyla were Proteobacteria and Bacteroidetes and BIO application enriched the genera Comamonas, Chitinophaga, the species Bacillus flexus and uncultured Bacillus. All the results showed that 2-year continuous application of BIO containing B. amyloliquefaciens NJN-6 more effectively controlled Fusarium wilt disease and improved fruit yields under field conditions and modulated banana rhizosphere microflora.     

A suite of complementary biocontrol traits allows a native consortium of root‐associated bacteria to protect their host plant from a fungal sudden‐wilt disease

The beneficial effects of plant‐–bacterial interactions in controlling plant pests have been extensively studied with single bacterial isolates. However, in nature, bacteria interact with plants in multitaxa consortia, systems which remain poorly understood. Previously, we demonstrated that a consortium of five native bacterial isolates protected their host plant Nicotiana attenuata from a sudden wilt disease. Here we explore the mechanisms behind the protection effect against the native pathosystem. Three members of the consortium, Pseudomonas azotoformans A70, P. frederiksbergensis A176 and Arthrobacter nitroguajacolicus E46, form biofilms when grown individually in vitro, and the amount of biofilm increased synergistically in the five‐membered consortium, including two Bacillus species, B. megaterium and B. mojavensis. Fluorescence in situ hybridization and scanning electron microscopy in planta imaging techniques confirmed biofilm formation and revealed locally distinct distributions of the five bacterial strains colonizing different areas on the plant‐root surface. One of the five isolates, K1 B. mojavensis produces the antifungal compound surfactin, under in vitro and in vivo conditions, clearly inhibiting fungal growth. Furthermore, isolates A70 and A176 produce siderophores under in vitro conditions. Based on these results we infer that the consortium of five bacterial isolates protects its host against fungal phytopathogens via complementary traits. The study should encourage researchers to create synthetic communities from native strains of different genera to improve bioprotection against wilting diseases.