ITS序列结合培养特征鉴定梨树腐烂病菌

对采自中国4个省份的9个梨树腐烂病菌分离株和7个苹果树腐烂病菌分离株的ITS序列进行了测定和分析,并结合GenBank的有性型Valsa ceratosperma、V.ambiens和V.mali的ITS序列构建了系统发育树。结果表明梨和苹果树的各分离株在ITS核苷酸序列上分化较小(p-distance=1.55%),均在V.ceratosperma聚类组,但二者又分别处于两个独立小分支。其与V.ambiens和V.mali处在不同的聚类组中,且亲缘关系较远,表明供试梨树腐烂病菌并非V.ambiens。培养性状和生物学特点的研究结果还发现,梨树腐烂病菌各分离株无论在菌落颜色、产孢特点、还是37℃高温的生长情况都和苹果腐烂病菌有一定差别。前者菌落始在PDA终为乳白色,而后者菌落初为白色后期变褐色;在20%ABA上,前者形成的产孢体较大而数量较少,在37℃高温下能正常生长,后者则形成的产孢体较小而数量较多,在37℃高温下不能正常生长。并未发现二者在子实体上有稳定明显的差异。因而表明梨树腐烂病菌应为V.ceratosperma,但可用培养性状和生物学特点进行区分其和苹果树腐烂病菌。     

苹果树腐烂病生防真菌Z-12A的鉴定及其生防效果

【背景】由苹果黑腐皮壳菌(Valsa mali)引起的苹果树腐烂病是我国苹果产区的毁灭性病害之一,具有分布广、危害重和防治难等特点,严重制约着苹果产业的发展,急需寻找一种对该病高效、安全的防治措施。【目的】明确一株分离自甘肃省静宁县苹果树根际土壤的生防菌株Z-12A的分类地位,评价其对苹果树腐烂病的生防潜力。【方法】结合形态学特征和rDNA ITS序列分析对拮抗菌株Z-12A进行鉴定,采用生长速率法测定了其生物学特性;采用平板对峙培养法和生长速率法测定菌株Z-12A对苹果树腐烂病菌菌丝生长的影响;利用菌株Z-12A活菌和发酵液分别处理苹果枝条及小白鼠测定其生物安全性;使用离体枝条烫伤接种法测定菌株Z-12A对腐烂病的防治效果。【结果】形态学观察和rDNA ITS序列分析结果表明该菌株为埃及青霉(Penicillium egyptiacum)。该生防菌菌丝生长的最适培养基是萨氏培养基(Sabouraud Dextrose Agar With Yeast Extract,SDAY),而高氏1号培养基(Gauze’s Medium No.1,GA)上产孢量最大;菌丝生长和产孢的最佳碳源是木糖,最佳氮源是硝酸钾;菌丝生长的最佳pH为5.0,最适宜产孢的pH为7.0;25℃时菌丝生长最快,产孢的最适温度为10℃。平板对峙试验表明菌株Z-12A对苹果树腐烂病菌抑制率为88.71%,发酵液在培养皿内的抑菌率为61.07%;显微观察显示菌株Z-12A可使苹果树腐烂病菌菌丝畸形及细胞原生质外渗;其发酵液对离体枝条防效为66.69%;安全性评价表明菌株Z-12A对苹果树和小白鼠安全。【结论】菌株Z-12A对苹果树腐烂病具有较强的生物防治效果,具有一定的生防潜能,可为该病生防菌剂的选择提供新的菌种资源。     

芽孢杆菌拮抗苹果树腐烂病菌的筛选、鉴定及抑菌活性初探

【背景】苹果树腐烂病发生面积广、危害严重,目前对于该病的防治以化学防治为主,但存在一定的弊端,因此迫切需要寻找一种对该病高效、安全的防治措施。【目的】从敦煌地区盐碱土壤中筛选出对苹果树腐烂病有良好抑制作用的芽孢杆菌菌株。【方法】采用平板对峙培养法和菌丝生长速率法对盐碱土中分离筛选的芽孢杆菌进行拮抗苹果树腐烂病菌的筛选,并对优良拮抗效果菌株进行离体组织防效测定、发酵培养基选择以及结合培养形态、生理生化及16S rRNA基因序列分析进行菌种鉴定。【结果】初筛阶段菌株7-2-1-2对病原菌的抑制率为86.18%,复筛试验中发酵液对病原菌的抑制率为73.2%;在离体枝条创面涂抹菌株7-2-1-2发酵原液后,苹果树腐烂病菌的生长可被完全抑制;菌株7-2-1-2的最优生长培养基为NBY培养基;经初步鉴定,菌株7-2-1-2为枯草芽孢杆菌亚种(Bacillus subtilis subsp. spizizenii)。【结论】菌株7-2-1-2对苹果树腐烂病有较好的生防效果,为该病生防菌剂的选择提供了必要的菌种资源。     

DNA sequence analysis of herbarium specimens facilitates the revival of Botrytis mali, a postharvest pathogen of apple

The fungus Botrytis cinerea has been widely accepted as the species responsible for causing gray mold decay of apple, although a second species causing apple decay, B. mali, was reported in 1931. Botrytis mali was validly published in 1931, nevertheless it has always been considered a doubtful species. To study the relationship of Botrytis isolates causing gray mold on apple, DNA sequence analysis was employed. Twenty-eight Botrytis isolates consisting of 10 species were sampled, including two B. mali herbarium specimens from apple originally deposited in 1932. The DNA sequence analysis of the beta-tubulin and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) genes placed the isolates into groupings with defined species boundaries that generally reflected the morphologically based model for Botrytis classification. The B. cinerea isolates from apple and other host plants were placed in a single clade. The B. mali herbarium specimens however always fell well outside that clade. The DNA sequence analysis reported in this study support the initial work by Ruehle (1931) describing the apple pathogen B. mali as a unique species.     

香梨内生拮抗细菌的筛选及对梨火疫病的生防潜力

该研究以健康香梨树的新鲜花器、当年生新生枝条、叶片和果实为试验材料,采用植物组织内生细菌分离法获得内生细菌菌株,培养菌落,并采用平板对峙法初筛和发酵液复筛对梨火疫病菌(Erwinia amylovora)、梢枯病菌(Pseudomonas syringae pv.syringae)和腐烂病菌(Valsa mali var.pyri)具有抑菌作用的细菌;将筛选获得的拮抗梨火疫病菌的菌株通过喷施香梨离体花序和盆栽杜梨苗测定其防病效果,以发掘内生细菌的生防资源,为探索梨树病害的生物防治途径奠定基础。结果显示:(1)从香梨枝条、叶、花和果实组织中共分离出337个内生细菌菌株,其中有14株具有明显的抑菌作用;进一步筛选出对梨火疫病菌、梢枯病菌和腐烂病菌具有抑菌作用的细菌分别有8株、9株和4株,其中ZN5、SN19和HN9菌株对梨梢枯病菌和腐烂病菌均有拮抗作用,HN89菌株对3种病原菌都具有较强的抑菌作用;共筛选出对梨火疫病菌具有抑菌作用的7个菌株(SN37、HN89、HN98、HN143、HN126、TN68、TN16)和具有竞争作用的TN50菌株。(2)内生菌株TN50预处理(喷施)对预防香梨花腐的效果显著,其防效(52.36%)与农用链霉素(60.67%)相近;HN89的防效(39.66%)其次;内生菌株TN50、HN89和SN37预处理(喷施)能够显著降低盆栽杜梨苗嫩枝的枝枯率和病情指数(P<0.05),7~15 d的平均保护性防效分别为67.20%、54.32%和45.91%,治疗性防效分别为63.88%、52.10%和36.17%。(3)结合形态特征和16S DNA序列分析,TN50、HN89和SN37菌株分别鉴定为克雷伯氏菌属(Klebsiella sp.)、类芽孢杆菌属(Paenibacillus sp.)和假单胞杆菌属(Pseudomonas sp.)。     

Delimiting cryptic pathogen species causing apple Valsa canker with multilocus data

Fungal diseases are posing tremendous threats to global economy and food safety. Among them, Valsa canker, caused by fungi of Valsa and their Cytospora anamorphs, has been a serious threat to fruit and forest trees and is one of the most destructive diseases of apple in East Asia, particularly. Accurate and robust delimitation of pathogen species is not only essential for the development of effective disease control programs, but also will advance our understanding of the emergence of plant diseases. However, species delimitation is especially difficult in Valsa because of the high variability of morphological traits and in many cases the lack of the teleomorph. In this study, we delimitated species boundary for pathogens causing apple Valsa canker with a multifaceted approach. Based on three independent loci, the internal transcribed spacer (ITS), β‐tubulin (Btu), and translation elongation factor‐1 alpha (EF1α), we inferred gene trees with both maximum likelihood and Bayesian methods, estimated species tree with Bayesian multispecies coalescent approaches, and validated species tree with Bayesian species delimitation. Through divergence time estimation and ancestral host reconstruction, we tested the possible underlying mechanisms for fungal speciation and host‐range change. Our results proved that two varieties of the former morphological species V. mali represented two distinct species, V. mali and V. pyri, which diverged about 5 million years ago, much later than the divergence of their preferred hosts, excluding a scenario of fungi–host co‐speciation. The marked different thermal preferences and contrasting pathogenicity in cross‐inoculation suggest ecological divergences between the two species. Apple was the most likely ancestral host for both V. mali and V. pyri. Host‐range expansion led to the occurrence of V. pyri on both pear and apple. Our results also represent an example in which ITS data might underestimate species diversity.     

PEG 介导的苹果腐烂病菌原生质体转化

摘要: 【目的】建立PEG 介导的苹果腐烂病菌原生质体遗传转化体系。【方法】本文利用带有hph 基因的质粒,以苹果腐烂病菌(Valsa mali var.mali) 03-8 为受体菌株,通过PEG 融合法对其原生体进行转化。【结果】于YEPD 内培养48 h 的菌丝,在酶解液浓度为50 mg /mL Driselase + 10 mg /mL Lysing Enzymes 情况下,按10 mL酶液/0. 5 g湿菌体比例,酶解2 h时可以释放出4 × 107 个/mL 原生质体,其转化效率为44 个/μg DNA。对转化子的PCR 检测和Southern 杂交分析表明,hph 基因已经整合进苹果树腐烂病菌的基因组中。转化子在PDA 培养基中继代5 次后,87. 5% 的转化子仍能正常生长,表明外源基因hph 能在苹果树腐烂病菌中稳定遗传。【结论】该转化体系的建立为苹果树腐烂病菌致病相关基因的深入研究奠定了基础。     

苹果树腐烂病菌不同致病力菌株对苹果的诱导效应

以‘富士’苹果叶片为材料, 采用刺伤接种法, 比较了苹果树腐烂病菌强致病菌LXS080601和弱致病菌LXS081501侵染对寄主体内丙二醛（MDA）含量、渗透调节物质及防御酶活性的影响。结果表明, 两菌株侵染后, 叶片MDA、蛋白质和可溶性糖含量均增加, 接种LXS080601叶片MDA含量快速上升, 最大增幅为141.15%, 而接种LXS081501叶片的MDA含量变化较小, 增幅仅为1.16%-16.24%, 但后者可溶性糖和蛋白质含量增幅分别高达158.12%和113.57%, 显著高于接种LXS080601的处理。同时, 两菌株均能诱导叶片内4种防御酶活性的升高, 但LXS081501诱导的苯丙氨酸解氨酶（PAL）、过氧化物酶（POD）、过氧化氢酶（CAT）和多酚氧化酶（PPO）活性均显著高于LXS080601, 说明不同致病力腐烂病菌对寄主防御酶活性的影响存在显著差异。     

Genetic Mapping Reveals Sophisticated Responses of Malus domestica to Botryosphaeria dothidea Isolates

Infection by the globally distributed pathogenic fungus Botryosphaeria dothidea (Moug.) Ces. et de Not. causes bot canker on the stems, branches and limbs or causes fruit ring rot (FRR) on the fruit in apple. To investigate the relationship between resistance to bot canker and FRR and among resistance mechanisms in response to different pathogen isolates, 34 major gene loci and six quantitative trait loci (QTLs) for bot canker and FRR resistance/susceptibility were genetically mapped on Malus genome using an F1 hybrid population (Jonathan × Golden Delicious) in 2008 and 2009. None of the QTLs for bot canker and FRR overlapped with the region of the major gene loci. Some of the FRR‐ and bot canker‐associated loci either coincided or overlapped, and some were independent, suggesting that the responses of different organs to the pathogen would be correlated but might deviate from each other. Some major gene loci and QTLs associated with different pathogen isolates of bot canker or, FRR were mapped to different regions of the genome. The results indicated that not only did the resistance mechanisms differ between bot canker and FRR but also that genetic differentiation occurred among pathogen isolates.     

Agrobacterium tumefaciens-Mediated Transformation of the Causative Agent of Valsa canker of Apple Tree Valsa mali var. mali

Valsa mali var. mali (Vmm), which is the causative agent of Valsa canker of apple tree, causes heavy damage to apple production in eastern Asia. In this article, we report Agrobacterium tumefaciens-mediated transformation (ATMT) of Vmm and expression of gfp (green fluorescent protein) in this fungus. The transformation system was optimized to a transformation efficiency of approximately 150 transformants/10⁶ conidia, and a library containing over 4,000 transformants was generated. The tested transformants were mitotically stable. One hundred percent hph (hygromycin B phosphotransferase) integration into Vmm was identified by PCR and five single-copy integration of T-DNA was detected in the eighteen transformants by Southern blot. To our knowledge, this is the first report of ATMT of Vmm. Furthermore, this library has been used to identify genes involved in the virulence of the pathogen, and the transformation system may also be useful to the transformation of other species of the genus Valsa.     

The apple FERONIA receptor-like kinase MdMRLK2 negatively regulates Valsa canker resistance by suppressing defence responses and hypersensitive reaction

Valsa canker, caused by the fungus Valsa mali, is one of the most destructive diseases of apple trees in China and other East Asian countries. The plant receptor-like kinase FERONIA is involved in plant cell growth, development, and immunity. However, little is known about the function of FERONIA in apple defence against V. mali. In this study, we found that MdMRLK2 was highly induced by V. mali in twigs of V. mali-susceptible Malus mellana but not in those of the resistant species Malus yunnaensis. 35S:MdMRLK2 apple plants showed compromised resistance relative to wild-type (WT) plants. Further analyses indicated that 35S:MdMRLK2 apple plants had enhanced abscisic acid (ABA) levels and reduced salicylic acid (SA) levels relative to the WT on V. mali infection. MdMRLK2 overexpression also suppressed polyphenol accumulation and inhibited the activities of phenylalanine ammonia-lyase (PAL), β-1,3-glucanase (GLU), and chitinase (CHT) during V. mali infection. Moreover, MdMRLK2 interacted with MdHIR1, a hypersensitive-induced response protein, and suppressed the MdHIR1-mediated hypersensitive reaction (HR), probably by impairing MdHIR1 self-interaction. Collectively, these findings demonstrate that overexpression of MdMRLK2 compromises Valsa canker resistance, probably by (a) altering ABA and SA levels, (b) suppressing polyphenol accumulation, (c) inhibiting PAL, GLU, and CHT activities, and (d) blocking MdHIR1-mediated HR by disrupting MdHIR1 self-interaction.     

Acremonium hansfordii,一中国新记录种及其抗植物病原真菌活性的研究

Acremonium hansfordii是中国一新记录种,在PDA平板上对多种植物病原真菌有很强的抑制作用。通过95%乙醇提取、YPR-Ⅱ型大孔吸附树脂吸附、乙酸乙酯萃取、沸点30-60℃石油醚沉淀等方法从Acremonium hansfordii菌丝体分离纯化出具有抗植物病原真菌活性的化合物枝顶孢素(acremonin),并得到结晶。acremonin对多种植物病原真菌都有抑制作用,其中对苹果腐烂病菌Valsa mali的IC50为0.095μg/mL,热稳定,121℃灭菌30min抑菌活性保留91.9%,pH4-9稳定。     

瑞拉菌素产生菌的鉴定

自陕西秦岭太白山土壤中分离到 1株编号为S 5 12 0的放线菌。根据对其生物特征鉴定、生理生化特征分析 ,它与链霉菌属中委内瑞拉链霉菌最为相近 ,但菌种S 5 12 0对梨黑星病菌、苹果腐烂病菌等多种引起植物病害的病原真菌有拮抗和溶菌作用 ,故认为S 5 12 0是委内瑞拉链霉菌的一个新变种 ,定名为委内瑞拉链霉菌秦岭变种(Streptomycesvenezuelaevar .qinlingensis.n .Var)。     

Studies on Apple Canker Disease. The Necrotic Toxins Produced by Valsa ceratosperma

Several metabolites responsible for the toxic manifestations of Valsa ceratosperma (Toda et Fries) Maire, a phytopathogenic fungus of the Japanese apple canker, have been isolated from its culture filtrate after growth on apple branch extract. Chemical and spectrometric studies revealed the products to be degradation products of phlorizin which is a dominant component distributed in leaves, stems, fruits and roots of apple. The toxic substances were identified as 3-(p-hydroxyphenyl) propionic acid, phloroglucinol, p-hydroxyacetophenone, p-hydroxybenzoic acid and protocatechuic acid. All of these compounds except p-hydroxyacetophenone were detected in the lesions of apple trees infected by V. ceratosperma. The fungus cultivated in a medium containing added phlorizin also produced the five toxic substances mentioned above. These results suggest that phlorizin is involved in the specific relationship between the host and the pathogen, indicating that the degradation products of phlorizin play important roles in the production of symptoms of infected apple trees.     

Characterization of Bacillus altitudinis as a New Causative Agent of Bacterial Soft Rot

A total of 67 bacterial isolates were obtained from apple and pear fruits with signs of soft rot collected from Egyptian markets. Pathogenicity tests showed that 25 isolates (37%) were pathogenic to apple and pear fruits, with considerable variation of virulence. Among these isolates, 16 (64%) were Gram‐positive, motile, spore‐forming long rods and were identified as members of the genus Bacillus based on an API test. In addition, five isolates (20%) were Gram‐negative, non‐spore‐forming, motile, oxidase and catalase‐positive short rods and were identified as members of the genus Pseudomonas. Furthermore, four isolates (16%) were Gram‐negative, non‐spore‐forming, motile, catalase‐positive and oxidase negative short rods and were identified as belonging to the genus Erwinia. All selected isolates showed a wide host range and could cause soft rot of all representative fruits and vegetables tested. The three most virulent isolates, AB4, AB6 and PB6, exhibited the highest soft rot severity on different apple and pear cultivars, and apple cv. Anna (116) was the most susceptible to infection by isolates AB4 and AB6, with soft rot severities of 63.33 and 60.67%, respectively. Also, pear cv. Le‐Conte was most susceptible to infection by isolate AB6, with a soft rot severity of 89.9%. A phylogenetic tree based on 16S rRNA gene sequences indicated that strains AB4 and AB6 were very similar to one another and also showed a similarity of 99% to Bacillus altitudinis, and strain PB6 revealed a similarity of 99% to Bacillus pumilus. To our knowledge, this is the first report of B. altitudinis as a soft rot pathogen for both apple and pear fruits.     

山西省苹果树腐烂病菌的种群结构分析

为明确山西省苹果树腐烂病菌的分布与组成,本研究采集并分离得到来自山西省8个苹果树种植区有代表性的78株腐烂病菌,通过形态特征观察及利用2个DNA片段（ITS和EF1α）构建系统发育树等方法对其遗传结构进行了研究。结果表明山西省苹果树腐烂病由苹果黑腐皮壳菌Valsa mali和梨黑腐皮壳菌Valsa pyri 2个致病种所致,其中V. pyri是优势种,分布于山西省各个苹果树种植区,占菌株总数的58.97%。以山西省8个苹果树种植区腐烂病菌各个种的相对频率进行聚类分析,欧式距离9作为聚类分割点将其种群结构分为3类。第Ⅰ类包括忻州、朔州2个苹果树种植区,V. pyri为该类型苹果树种植区的致病菌、相对频率为100%。第Ⅱ类包括晋城、长治、临汾、太原、运城5个苹果树种植区,V. pyri是这5个种植区的优势种。第Ⅲ类包含晋中1个苹果树种植区,V. mali是该种植区的优势种。     

Transcriptomic Analysis Revealed Hormone-Related and Receptor-Like Kinase Genes Involved in Wound Healing of ‘Duli’ and its Resistance to Valsa Pyri

Plant Molecular Biology Reporter - Valsa canker is a destructive fungal disease that results in a serious loss of production. The pathogen Valsa pyri (Vp) usually infiltrates the bark and xylem via...     

新疆野果林苹果腐烂病病原菌鉴定及药用植物内生细菌对其抑菌效果

【背景】药用植物内生细菌能产生与寄主植物相同或相似的化合物及一些新的次级代谢产物等,具有促进宿主植物生长、抵抗病虫害、降解有毒有害化合物等作用。【目的】进一步提高苹果腐烂病生物防治的效率,丰富新疆药用植物内生细菌拮抗功能菌株的资源库。【方法】从新疆伊犁新源县和塔城额敏县野果林中采集带腐烂病病斑的果树枝条,分离鉴定苹果腐烂病病原菌,并采用平板对峙法从药用植物内生细菌中筛选对苹果腐烂病具有抑制作用的拮抗菌株。【结果】从两地共分离获得234株分离株,筛选鉴定出25株Valsa malicola和2株Valsa mali;同时,筛选出92株具有抑菌效果的内生细菌菌株,其中70株来自甘草植物内生细菌。【结论】药用植物甘草中富含较为丰富的抗苹果腐烂病病原菌的微生物菌株资源。本研究在新疆野果林苹果腐烂病的生物防治及药用植物内生细菌的开发利用等方面具有重要意义。