束状刺盘孢的体外培养条件研究

目的对束状刺盘孢体外培养,观察形态特征。方法将束状刺盘孢接种在马铃薯葡萄糖琼脂培养基(PDA)中分别置于4℃、28℃、35℃和37℃下培养2周,观察生长情况;选取生长最好的菌株分别接种在沙堡弱培养基(SDA)、PDA、察氏培养基(CPA)、胡萝卜琼脂培养基(CDA)和玉米粉培养基(CMA)中,同一温度下培养2周,观察菌落形态及镜下形态。结果菌株在28℃条件下生长最快、菌落发育饱满、产生灰色色素;菌株在五种培养基中生长快慢依次是PDA>CDA>CMA>SDA>CPA,菌落在PDA、CDA和CMA中呈鼠灰色,SDA中呈白色和棕色,CPA中呈白色和鼠灰色,SDA和CPA中未见分生孢子和刚毛产生。结论在PDA、CDA和CMA中28℃条件下,较适合束状刺盘孢生长。     

石楠拟盘多毛孢毒素产生条件初探

石楠拟盘多毛孢是引起草莓根腐病的优势病原菌之一, 本实验在明确该病菌毒素是主要致病物质的基础上, 利用叶圆片法对该菌的产毒条件进行了初步探讨, 结果表明, 除光照影响不明显外, pH值、温度、振荡及培养时间等对此菌产生毒素均有显著影响, 其最适产毒条件为：培养基初始pH值为自然pH值(约为6.2)、温度为25°C、黑暗、静置培养5 d~7 d。此外, 实验还发现该菌产生的粗毒素对玉米、黑麦和绿豆种子萌发、根或芽的延伸生长都有明显的抑制作用。     

对进境高粱种子中高粱炭疽病菌疏纹刺盘孢的检疫

从一批进境的美国高粱种子样品中分离得到一株与疏纹刺盘孢Colletotrichum sublineola形态相似的菌株6222-1。经形态学观察发现该菌产生大量镰状或梭形分生孢子,在菌丝末端形成膨大的附着胞。经多位点基因(ITS、GAPDH、CHS-1、HIS3、ACT、TUB2)系统发育分析,发现该菌株与C.sublineola模式菌株聚集在同一个分支上,支持率达到100%。经病原菌接种高粱幼苗叶部,引起典型的炭疽病症状。根据上述研究结果,确认该菌株为疏纹刺盘孢。这是我国首次截获该危险性病原真菌。     

刺盘孢属及其近似属的五个中国新记录种

报道我国5个新记录种:榕刺盘孢 Colletotrichum ficus Koord.、印度胶树刺盘孢Colletotrichum fici-elasticae、卫矛痂圆孢Sphaceloma euonymi-jasponici、茼蒿柱盘孢Cylindrosporium chryanthemi和楤木Gloeosporium araliae.研究标本保存于云南农业大学真菌标本室(MHYAU)和西北农林科技大学真菌标本室(FHNWAC).     

抗辣椒炭疽病菌盘长孢状刺盘孢的木霉菌的筛选

本试验通过对峙培养法筛选出28株具有强拮抗能力和15株中等拮抗能力的木霉菌。使用玻璃纸培养法进一步复筛出6株木霉菌(104、393、424、402、451和123T),其非挥发性次生代谢产物对盘长孢状刺盘孢的抑制率高达92%以上。通过平板对扣培养法发现木霉菌424(Trichoderma atroviride)以及123T (T. paratroviride)的挥发性次生代谢产物对盘长孢状刺盘孢也具有抑制作用,抑制率分别为57.12%和60.43%。试验还对9个辣椒品种进行了盘长孢状刺盘孢的果实致病性评价,发现红尖椒属于高度易感病品种。在对该品种进行针尖刺伤法处理后,喷施木霉菌424和123T的分生孢子悬浮液后可以提高其对病菌的抗性。此外,试验还发现上述2株木霉菌的孢子悬浮液对辣椒种子萌发及幼苗生长具有促进作用,其将辣椒种子的发芽率由38.9%提高至70.0%。同时,辣椒幼苗经木霉菌分生孢子处理后,株高、根长、茎粗、鲜重和干重分别增加了52.5%、96.0%、104.0%、372.0%和224.0%。综上所述,本试验筛选的木霉菌424和123T能够充分发挥其生防能力,为进一步开发辣椒炭...     

束状刺盘孢角膜炎的实验研究

目的初步研究束状刺盘孢感染兔角膜的临床表现和组织病理学特征。方法选择15只新西兰白兔,应用基质内直接注射法建立模型。肉眼观察角膜变化,并在接种后第5 d、14 d、21 d处死家兔,对角膜行HE染色和PAS染色,观察其组织病理学改变。结果兔束状刺盘孢角膜炎动物模型成功建立,肉眼可以见到典型的真菌性角膜炎的表现。组织病理学检查,在感染早期即可看到大量真菌病原体生长,菌丝垂直于角膜基质,后期出现新生血管和瘢痕。结论角膜基质内直接注射法复制真菌性角膜炎动物模型成功率高、稳定。兔束状刺盘孢角膜炎有典型的真菌性角膜炎的表现,组织学中菌丝垂直于角膜基质生长。     

苹果炭疽叶枯病病原学研究

炭疽叶枯病菌能够侵染苹果叶片造成病叶早期干枯、脱落,侵染果实引起坏死性斑点。该病害近年来在我国一些苹果产区被发现,并有迅速蔓延的趋势。对采自河南和陕西的苹果炭疽叶枯病的病原菌进行了形态学、培养特性、致病性及分子系统发育研究,明确了引起该病害的病原为果生刺盘孢Colletotrichum fructicola和隐秘刺盘孢C.aenigma。经致病性测定,证明C.fructicola和C.aenigma对嘎拉、秦冠、金冠、粉红女士、太平洋玫瑰、金世纪、蜜脆等以金冠为亲本的品种叶片致病;在有伤接种时,C.fructicola和C.aenigma对嘎拉、金冠、秦冠、太平洋玫瑰、新红星、富士等品种果实具有致病性;在无伤接种时,C.fructicola对嘎拉、金冠果实致病,C.aenigma对嘎拉果实致病。研究结果表明,C.fructicola和C.aenigma对不同品种叶片和果实的致病性都存在明显的分化现象。     

瓜类刺盘孢诱导物对新疆甜瓜抗病相关酶活性的影响

近年来,应用病毒、细菌、真菌以及病原菌体及其代谢物作为诱导因子,已在多种作物上获得对病害的整体免疫,有些已开始在田间应用。我们也曾应用人工诱导免疫的方法使新疆甜瓜获得对瓜类疫霉病的抗性,但有关植物人工诱导免疫机理的研究,目前国外报道不多,国内尚未见报道。本文以瓜类刺盘孢(Colletotrichum langenarium)培养滤液和菌丝细胞壁作为诱导物,研究了免疫植株相关酶的活性以及可溶性蛋白的变化,探讨了人工诱导免疫的机理。     

长蠕孢菌产孢条件的研究

对番茄褐斑病原菌—长蠕孢菌产孢条件进行了研究,结果表明燕麦片琼胶培养基、Czapek培养基和PDA＋番茄叶片能促进产孢子,V8汁、PSA和番茄汁培养基抑制产孢;碳源果糖明显促进产孢,甘露醇抑制产孢子;氮源氯化铵促进产孢,蛋白胨和硫酸铵抑制产孢;光照和紫外线照射对长蠕孢菌产孢有明显促进作用,特别是紫外线照射60～80min时产孢量达到最大;偏低温或偏高温以及微碱性环境能促进长蠕孢菌产孢,温度为15℃或30℃,pH8～9时最有利于产孢。     

Fungal effector SIB1 of Colletotrichum orbiculare has unique structural features and can suppress plant immunity in Nicotiana benthamiana

Fungal plant pathogens secrete virulence-related proteins, called effectors, to establish host infection; however, the details are not fully understood yet. Functional screening of effector candidates using Agrobacterium-mediated transient expression assay in Nicotiana benthamiana identified two virulence-related effectors, named SIB1 and SIB2 (Suppression of Immunity in N. benthamiana), of an anthracnose fungus Colletotrichum orbiculare, which infects both cucurbits and N. benthamiana. The Agrobacterium-mediated transient expression of SIB1 or SIB2 increased the susceptibility of N. benthamiana to C. orbiculare, which suggested these effectors can suppress immune responses in N. benthamiana. The presence of SIB1 and SIB2 homologs was found to be limited to the genus Colletotrichum. SIB1 suppressed both (i) the generation of reactive oxygen species triggered by two different pathogen-associated molecular patterns, chitin and flg22, and (ii) the cell death response triggered by the Phytophthora infestans INF1 elicitin in N. benthamiana. We determined the NMR-based structure of SIB1 to obtain its structural insights. The three-dimensional structure of SIB1 comprises five β-strands, each containing three disulfide bonds. The overall conformation was found to be a cylindrical shape, such as the well-known antiparallel β-barrel structure. However, the β-strands were found to display a unique topology, one pair of these β-strands formed a parallel β-sheet. These results suggest that the effector SIB1 present in Colletotrichum fungi has unique structural features and can suppress pathogen-associated molecular pattern–triggered immunity in N. benthamiana.     

Establishment of a selection marker recycling system for sequential transformation of the plant-pathogenic fungus Colletotrichum orbiculare

Genome sequencing of pathogenic fungi has revealed the presence of various effectors that aid pathogen invasion by the manipulation of plant immunity. Effectors are often individually dispensable because of duplication and functional redundancy as a result of the arms race between host plants and pathogens. To study effectors that have functional redundancy, multiple gene disruption is often required. However, the number of selection markers that can be used for gene targeting is limited. Here, we established a marker recycling system that allows the use of the same selection marker in successive transformations in the model fungal pathogen Colletotrichum orbiculare, a causal agent of anthracnose disease in plants belonging to the Cucurbitaceae. We identified two C. orbiculare homologues of yeast URA3/pyrG, designated as URA3A and URA3B, which can be used as selection markers on medium with no uridine. The gene can then be removed from the genome via homologous recombination when the fungus is grown in the presence of 5-fluoroorotic acid (5-FOA), a chemical that is converted into a toxin by URA3 activity. The ura3a/b double mutants showed auxotrophy for uridine and insensitivity to 5-FOA. Using the ura3a/b mutants, transformation with the URA3B marker and its removal were successfully applied to disrupt the virulence-related gene, PKS1. The pks1 mutants showed a reduction in virulence, demonstrating that the method can be used to study virulence-related genes in C. orbiculare. The establishment of a URA3-based marker recycling system in plant-pathogenic fungi enables the genetic analysis of multiple genes that have redundant functions, including effector genes.     

New report of Colletotrichum gloeosporioides causing anthracnose of Pisonia alba in India

Anthracnose was observed on Pisonia alba plants as irregular, black, necrotic spots that often coalesce to form large necrotic area on leaves. A fungus, consistentlyisolated from symptomatic leaves was identified as C. gloeosporioides on the basis of morphological and cultural characteristics. The fungus produced white mycelia, which became dark grey with later formation of numerous salmon pink coloured spore masses. The conidia were hyaline, unicellular, aseptate and oval to cylindrical with rounded ends and were 10–20 μm long and 3–5 μm wide. Pathogenicity tests conducted on healthy detached leaves of Pisonia plants showed typical anthracnose symptoms afterfour to seven days. This is the first report of anthracnose of Pisonia alba.     

Detection and molecular characterization of carbendazim-resistant Colletotrichum truncatum Isolates causing anthracnose of soybean in Thailand

A loss of fungicide efficacy, particularly for carbendazim, was noted in soybean fields in Thailand and was considered to be due to the development of Colletotrichum truncatum resistance. The carbendazim sensitivity of C. truncatum populations isolated from various soybean fields in Thailand was thus evaluated with in vitro sensitivity assays and molecular characterization of mutations in the sequences of the ß₂-tubulin (TUB2) gene that confer carbendazim resistance in the pathogen. Among 52 isolates, 46 isolates were classified as highly resistant (HR) to carbendazim (EC₅₀ > 1,000 µg/ml). All HR isolates grew on PDA amended with carbendazim at 1,000 µg/ml. Six isolates were classified as carbendazim sensitive (S) (EC₅₀ < 1 µg/ml). Mycelial growth on PDA amended with 1 µg/ml carbendazim was inhibited by over 50% compared with growth on PDA alone. When a partial TUB2 gene from the isolates was amplified and analysed using predicted amino acid sequences, an alteration from glutamic acid to alanine at codon 198 (E198A) was found in 45 HR isolates for which the EC₅₀ was higher than 2000 µg/ml. This mutation resulted from a nucleotide substitution from adenine to cytosine (GA G → GC G). The other HR isolate, CtPhS_1, with EC₅₀ of 1,127 µg/ml, had an alteration at codon 200 (F200Y) (TT C → TA C).     

In vitro and in vivo antagonism of biocontrol agents against Colletotrichum lindemuthianum causing bean anthracnose

Bean anthracnose caused by Colletotrichum lindemuthianum is a serious seed borne disease. For devising an effective management strategy, the efficacy of different bioagents, viz. Trichoderma viride, Trichoderma harzianum, Trichoderma hamatum and Gliocladium virens conducted under in vitro and in vivo conditions revealed maximum inhibition of mycelial growth in dual culture (59.48%) and inverted plate (55.98%) with T. viride. All the bioagents overgrew the pathogen and the principal mechanism of mycoparisitism observed was coiling, brusting and disintegration of pathogen hyphae. Culture filtrate from T. viride was found best as it completely inhibited radial growth at 25 and 50% concentration and reduced the spore germination of test fungus significantly. However, lower concentrations of culture filtrate from all bioagents showed little effect on spore germination. Seed application of bioagents was found better as compared to soil application. A maximum increase in seed germination and inhibition of seed borne infection was observed with T. viride followed by T. harzianum under pot culture conditions. T. viride has the maximum potentiality to suppress the spore germination, mycelial growth, seed borne infection of C. lindemuthianum and increased seed germination when compared with the other biocontrol agents.     

Management of postharvest disease of mango anthracnose incited by Colletotrichum gleosporioides

Abstract

Among the seven fungal (Gliocladium virens, Trichoderma hamatum, T. harzianum, T. koningii, T. longibrachiatum, T. pseudokoningii and T. viride) and two bacterial (Bacillus subtilis and Pseudomonas fluorescens) antagonists screened against C. gloeosporioides under in vitro conditions, T. harzianum exhibited maximum inhibition followed by Pseudomonas fluorescens at 5 days after incubation. These fungal and bacterial antagonists were selected for application to fruits infected with pathogens. Fruits inoculated with C. gloeosporioides were dipped in spore/cell suspensions of fungal/bacterial antagonists and kept for different durations. The fungal antagonists T. harzianum and P. fluorescens were effective in checking the spread of pathogens on fruits compared with the pathogen-inoculated control.     

PCR-based detection and characterization of the fungal pathogens Colletotrichum gloeosporioides and Colletotrichum capsici causing anthracnose in papaya (Carica papaya l.) in the Yucatan peninsula

Colletotrichum gloeosporioides is the common causal agent of anthracnose in papaya (Carica papaya L.) fruits, and infection by this fungal pathogen results in severe post-harvest losses. In the Yucatán peninsula (Mexico) a different Colletotrichum species was isolated from papaya fruits with atypical anthracnose lesions. The DNAs from a variety of Colletotrichum isolates producing typical and atypical lesions, respectively, were amplified by PCR with C.gloeosporioides-specific primers. All isolates from typical anthracnose lesions yielded a 450 bp PCR product, but DNAs from isolates with atypical lesions failed to produce an amplification product. For further characterization, the rDNA 5.8S-ITS region was amplified by PCR and processed for sequencing and RFLP analysis, respectively, to verify the identity of the papaya anthracnose pathogens. The results revealed unequivocally the existence of two Colletotrichum species causing anthracnose lesions on papaya fruits: C. gloeosporioides and C. capsici. PCR-RFLP using the restriction endonuclease MspI reliably reproduced restriction patterns specific for C. capsici or C. gloeosporioides. The generation of RFLP patterns by MspI (or AluI or RsaI) is a rapid, accurate, and unequivocal method for the detection and differentiation of these two Colletotrichum species.