The pecan tree [Carya illinoinensis (Wangenheim) K. Koch] produces seeds with high nutritional value and medicinal properties. Brown spot is one of the most important diseases of pecan in China, especially in Jiangsu province. Disease incidence ranged from 10% to 20% in the affected fields, which seriously affected the yield and quality of fruits. In this study, by tissue separation method, two Colletotrichum-like isolates were isolated from diseased pecan in different planting areas in Jurong. In order to identify these isolates, typical morphological characteristics were analyzed, phylogenetic tree was constructed based on ITS-TUB2-GAPDH- CHS-1-ACT genes, and their pathogenicities were tested. The results showed that ten strains were pathogenic and identified as C. fructicola and C. viniferum. Pathogenicity tests showed that all of the isolates could induce leaf necrosis, displaying variable pathogenicites for different strains. This is the first report of C. fructicola and C. viniferum causing pecan blackspot in Jurong, Jiangsu province, which will provide theoretical basis for further study of disease monitoring and control and breeding of disease resistance in pecan cultivars.

Anshun is the main producing area of yam (Dioscorea oppositifolia) in Guizhou Province. Recently, field investigations found that yam in Anshun area was suspected to be infected with cucurbit chlorotic yellows virus (CCYV), which caused huge economic losses to local farmers. The leaves suspected of being infected with CCYV were collected. RT-PCR detection was performed on the suspected diseased leaf samples, and CCYV was determined in combination with the sequencing results. Phylogenetic analysis was performed by CCYV coat protein (CP) sequence. The results showed that the isolates detected by Anshun in Guizhou had small regional differences with the isolates in USA and Saudi Arabia and other countries, and the relatives were relatively close, while the isolates in Iran was relatively distant. The findings will facilitate further research and help develop strategies for the prevention and control of CCYV in yam.

The avocado (Persea americana) is a commercially valuable fruit crop and cultivated in tropical region. Leaf anthracnose is a common disease of avocado. In this study, we isolated and identified a Colletotrichum strain from leaf spot disease of avocado at the nursery of Guangxi, China. According to the morphological and phylogenetic analyses of multi-locus sequences (internal transcribed spacer, ITS; chitin synthase, CHS-1; actin, ACT; glyceraldehydes-3-phosphate dehydrogenase, GAPDH), the pathogen was identified as Colletotrichum fructicola. To our knowledge, this is the first report of C. fructicola from leaf anthracnose on P. americana in China. This finding will help us to recognize the leaf anthracnose disease of avocado and establish a foundation for future studies on C. fructicola to address effective management strategies.

Alocasia odora (Araceae), native to South America, is a common perennial herb and horticultural plant in the tropical and subtropical regions. From July 2020 to May 2021, a leaf spot disease was found on A. odora on Yongxing island of Sansha City, Hainan Province, China. Disease symptoms such as brown lesions were observed on the leaf margins of the plants. Pathogenicity of the isolate was confirmed with Koch’s postulates. Based on morphological characteristics and phylogenetic analysis of ITS region, EF1-α and CAM gene sequences, the fungal isolate was identified as Fusarium guilinense. This is the first report of F. guilinense causing A. odora leaf spots in China.

In 2017, 2018 and 2020, a total of 191 leaf spot samples of maize were collected from representative sites of Beijing, Shandong and Jilin in Northern China. There were 293 strains -screened as Altarnaria sp. and 92 strains as Curvularis sp. based on rDNA ITS sequences analysis and morphological characteristics. Ten represen-tative strains of Alternaria were belonged to A. alternate by phylogenetic analysis with a combined dataset of rDNA-ITS, GAPDH, Tef1 and Alt a 1 gene fragments. Likewise, six representive strains of Curvularia were classified into C. lunata, C. intermedia and C. spicifera based on rDNA-ITS and GAPDH sequences. The pathogenicity by in vitro leaf assays were conducted for strains isolated from 102 samples and 42 samples of Alternaria and Curvularia, respectively, while by spraying with spore suspension were conducted for 20 representative strains. The result showed that all the strains mentioned above from four fungal species caused spot symptoms on maize leaves with the varied pathogenicity. In this study, the isolation frequency of Alternaria alternata and Curvularia lunata was significantly higher than that of C. intermedia and other species.

小麦条锈病是由小麦条锈菌引起的气传性真菌病害,在全球小麦种植区广泛发生,严重威胁小麦的安全生产。充分利用寄主抗病性是防治条锈病最经济的方法。富含亮氨酸重复类受体蛋白激酶 (leucine-rich repeat receptor-like protein kinase, LRR-RLK) 作为RLK家族最大分支的模式识别受体,在防御病菌入侵方面发挥重要作用。本研究系统鉴定了小麦LRR-RLKs家族成员,从小麦与条锈菌互作的转录组数据库中筛选出了43个显著差异表达的LRR-RLKs,最后选取了在亲和与非亲和互作过程中都显著上调表达的TaRLK3D.2为对象,利用实时定量PCR、亚细胞定位及大麦条纹花叶病毒介导的基因沉默初步研究了该基因在小麦与条锈菌互作过程中的功能。结果表明TaRLK3D.2定位于植物细胞膜,瞬时沉默TaRLK3.2后,条锈菌侵染严重度显著下降,生长发育变慢。作为典型的LRR-RLKs家族成员,TaRLK3D.2在小麦条锈菌侵染过程中可能行使感病功能。本研究初步明确了该基因的功能,以该基因为靶标通过基因编辑或诱变获取稳定的功能缺失突变体可创制持久抗条锈病材料,为生产服务。

苹果锈果类病毒(apple scar skin viroid,ASSVd)侵染引起苹果花脸病和锈果病,近年来对我国苹果产业造成了严重损失,但造成两种症状差异的分子特性尚不明确。本研究前期从我国山东和山西收集表现花脸和锈果症状的苹果果实样品,进行了克隆和测序,获得56个ASSVd分子变种的全长核苷酸序列,长度在330～333 nt之间。通过对本研究获得的ASSVd分子变种比对分析及与报道的症状相关分子变种间的比较,表明花脸和锈果症状的ASSVd分子变种之间的一致率为86.9%～100%,变异率为0.0%～13.3%,均有4个主要变异区域(含21个变异位点),位于致病(P)区、P区与左端区(T_L)邻接区域及P区与中央保守(C)区的邻接区域,二级结构呈紧凑的棍棒状。选取我国症状相关及国内外其他代表性ASSVd分子变种进行进化分析,显示我国ASSVd分离物分为7个簇(I～VII),且两种不同症状相关分子变种聚在相同簇(I、III和VI)。其中,ASSVd簇Ⅰ为优势组群,含有我国新疆及其他地区和希腊及其他国家来源的分离物,推测为原始组群;簇II、IV、V和VII表现出明显的我国地域专化性,推测为我国分离物的特有进化组群。对比本研究和国内外已报道两种症状相关ASSVd分子变种的核苷酸序列的长度、变异规律和进化关系,显示两种来源分子变种没有明显差异性,表明苹果两种症状之间的差异与ASSVd的分子变异没有相关性,而与苹果品种有关。研究结果为深入认识苹果花脸病和锈果病的病原分子特性及采用现代分子技术防治该病害提供了重要信息。

In order to clarify geminiviruses causing Bolbostemma paniculatum leaf shrinkage and mosaic in Anguo botanical garden, Hebei Province, China, the virus genome sequence was amplified by geminivirus degenerate primers and specific primers, and the phylogenetic tree of virus genome was constructed. The results showed that the squash leaf curl china virus (SLCCNV) was detected in Bolbostemma paniculatum plants in Hebei Province. The SLCCNV infected plants showed leaf shrinkage and mosaic, and the isolate was named SLCCNV-HB (DNA-A GenBank accession No: OL743525; DNA-B GenBank accession No: OM238222). The virus DNA-A genome was 2 736 bp and contained six open reading frames. The virus DNA-B genome was 2 718 bp and contained 2 open reading frames. The SLCCNV-HB DNA-A sequence shares the highest identity (96.30%) with SLCCNV-GDHY Pumpkin isolate (GenBank accession No: MW389917). The SLCCNV-HB DNA-B sequence shares the highest identity (95.37%.) with SLCCNV-GDHY Pumpkin isolate (GenBank accession No: MW389918). To our knowledge, this is the first report of SLCCNV that infects medicinal plant Bolbostemma paniculatum.

植物病毒病对世界农业生产构成严重威胁,保护粮食作物不受病毒侵害是农业的一个重大挑战。由分子生物技术主导的抗病毒工程已成为预防和控制植物病毒的有效策略。近期,CRISPR/Cas基因编辑系统介导的靶向DNA或RNA的进展,使其成为适用于植物保护领域极具吸引力的工具,正在被广泛用于生产抗病毒的栽培作物品种。本综述回顾CRISPR/Cas系统的发展,总结它们通过针对病毒序列或寄主易感基因控制不同植物病毒的应用;列出一些可用于抗病毒育种的潜在隐性抗性基因,并强调基于隐性抗性基因的抗病毒育种对产生无发育缺陷的转基因植物的重要性;描述CRISPR/Cas技术在田间检测和诊断植物病毒方面的应用。这一新兴分子技术的研究成果可以为扩展农业和粮食安全的发展作出重大贡献。

由苹果黑腐皮壳(Valsa mali)侵染引起的苹果树腐烂病是严重威胁我国苹果产业健康发展的重大枝干病害。揭示病菌致病机理有助于制定病害防控新策略。含cupin结构域蛋白是一个大的蛋白家族,广泛参与植物发育和逆境胁迫响应等多种生命活动。有关植物病原真菌中该类蛋白的研究报道很少,其参与菌丝生长和侵染致病的生物学功能尚不明确。基于V. mali与苹果树皮互作的转录组分析,发现一个在病菌侵染过程中显著上调表达的基因。蛋白序列特征分析发现,该蛋白含有1个典型的cupin结构域,且与其他物种含cupin结构域蛋白高度同源,将之命名为Vmcupin1。实时荧光定量分析发现Vmcupin1在病菌侵染过程中显著上调表达。创制了Vmcupin1缺失突变体和回补菌株,发现该基因缺失后,病菌生长速率在一定程度上降低,其致病力和适应H₂O₂和NaCl胁迫的能力显著降低,表明Vmcupin1在病菌营养生长、侵染致病,以及逆境胁迫中发挥重要功能。研究结果丰富了真菌含cupin结构域蛋白功能的认知,有助于全面解析腐烂病菌致病机理。

由炭疽菌(Colletotrichum gloeosporioides)引起的荔枝炭疽病是产业中最严重的病害之一,其周年发生,可以侵染叶片、花穗和果实。为评估荔枝资源的炭疽病抗病性、筛选抗性种质,本研究利用离体叶片,分析了接种方法、叶片叶龄、接种位置、发病时间和病斑测量方法对抗病性评价的影响。结果表明,选用叶片完全舒展、达到最大叶面积、刚完成转色的叶片,在叶片背面针刺接种炭疽菌菌饼(直径5 mm),接种后72 h观察发病情况,并测量病斑面积,能够较好的评估荔枝的抗病性。利用此方法分析了55份荔枝资源接种炭疽菌后的叶片病斑面积,利用欧式平方距离法将病情指数分为6级,根据发病情况将抗性等级归类为免疫、高抗、中抗、中感和高感。本研究建立了利用荔枝离体叶片评价抗炭疽病的技术方法,明确了评价标准,为荔枝抗病品种的选育、培育和抗病基因的筛选提供了技术支持。

Hippophae rhamnoides is a perennial shrub mostly used for water and soil conservation, wind break and sand fixation in western China. The fungal trunk canker disease on Hippophae rhamnoides is sometime very destructive in field. The authors isolated a pathogenic fungus strain A-1-2 from the diseased host plants in Erdos, Inner Mongolia. It was identified as Diaporthe eres by morphological and molecular methods. The pathogen grows fast on PDA, reaching 8 cm in diameter in 7 d. The most favorable temperature for mycelial growth was found at 25 ℃. Light was contributive to mycelial growth, while alternative light and dark was inducible to pycnidium formation. This paper is the first report for the pathogen Diaporthe eres causing trunk canker on H. rhamnoides in Inner Mongolia. The research results provide a preliminary base for further study on the disease cycle and management.

2019—2020年,在河南省进行病害调查时发现了小麦新病害茎基褐腐病,小麦灌浆期河南省周口市项城的发病率为35%,安阳市、漯河市和南阳市零星发病。通过对变褐叶鞘进行组织分离,得到74株白色稀疏绒状真菌分离物。基于多基因(ITS、β-tublin、RPB2和EF-1α)序列分析构建的系统发育树分析和菌株形态鉴定结果表明:74株分离菌株中22株为Microdochium nivale,52株为M. majus。M. nivale分生孢子呈镰刀状,大小为(11.62~26.04) μm ×(2.43~4.44)μm,平均为17.07 μm ×3.57 μm(n=240),有1~3个隔;M. majus分生孢子呈镰刀状,大小为(15.63~33.82) μm ×(3.36~8.95)μm,平均为25.14 μm ×5.45 μm(n=240),有1~6个隔。M. nivale和M. majus两种菌株最适宜的生长温度均为20 ℃,其在马铃薯葡萄糖琼脂培养基(PDA)平板上的平均生长速度分别为0.93 和0.89 cm·d^-1。M. nivale在5~25 ℃下的平均生长速度均高于M. majus。M. nivale和M. majus 在10 ℃条件下(PDA培养基)培养40 d后,均能产生大量分生孢子团,而其他温度产生的分生孢子团较少。代表性菌株菌丝体接种‘矮抗58'的结果表明,M. nivale和M. majus对‘矮抗58'发病率均为100%,病情指数分别为14.5~33.0和11.6~28.0,两种菌的致病力相当。本研究首次发现在河南省南阳、安阳、周口和漯河,M. majus和M. nivale可以引起小麦新病害即茎基褐腐病,研究结果为小麦茎基褐腐病监测提供理论依据。

为明确福建茶树潜隐病毒(camellia cryptic virus 1, CCV1)发生情况,探明其全基因组序列特征及其与油茶潜隐病毒1(camellia oleifera cryptic virus 1, CoCV1)的关系,本研究通过RT-PCR方法对采自福建7个茶树种植区疑似感染CCV1的叶片进行检测,并随机选取18个CCV1分离物,经扩增、克隆获得全基因组序列,对其序列特征和系统发育关系进行分析。结果显示,431份样品中共有94份样品检测出CCV1,检出率为21.81%。测定获得的18个CCV1分离物dsRNA1、dsRNA2和dsRNA3的核苷酸序列全长分别为1 722-1 725 bp、1 504-1 506 bp和1 497-1 499 bp。克隆获得的RdRp、CP和mCP的氨基酸序列与CCV1代表分离物Won(GenBank登录号MH898482-MH898484)和CoCV1代表分离物PXCS5(GenBank登录号MH814756-MH814758)的一致性值分别≥91%、≥84%和≥90%,超过了δ-分体病毒属(Deltapartitivirus)中种的界定标准阈值。进一步的系统发育分析结果显示,所有CCV1分离物与CoCV1 分离物以高置信值相聚成簇,证实CCV1和CoCV1为Deltapartitivirus属的同一种病毒。

In germplasm resource nursery of Guangxi South Subtropical Agricultural Science Research Institute, a stem disease of Baccaurea ramiflora was found at the seedling stage. The pathogen was determined by the Koch's postulates, morphological observation and ITS-TEF-SSU-LSU sequence analysis, and was identified as Athelia rolfsii. To our knowledge, this is the first report on A. rolfsii causing southern blight on B. ramiflora in China as well as in the world.

陕西省柞水、石泉、镇安县等地的华中五味子种植业,已成为当地乡村振兴的支柱产业。随着人工种植面积的不断扩大,其根部腐烂病十分突出,严重影响华中五味子产业发展。为明确引起华中五味子根腐病的病原,本研究从陕西省商洛市的柞水县、镇安县和宝鸡市的陇县五味子种植基地采集病株标本,经组织分离法和致病性检验获得华中五味子根腐病病原菌,并根据形态学以及ITS-TEF-1α-β-Tubulin序列串联分析,将其鉴定为层出镰刀菌(Fusarium proliferatum),杀菌剂室内抑菌活性测定结果表明,供试11种杀菌剂中吡唑醚菌酯、叶菌唑、咯菌腈和戊唑醇对其抑菌作用最强,EC₅₀值分别为0.021 0、0.083 3、0.380 7和0.403 5 μg·mL^-1。本研究为陕西地区华中五味子根腐病的有效防控提供重要指导依据。

Strawberry root rot is one of main diseases on strawberry in Shandong Province. In recent years, a new root rot was observed on strawberry in Jinan and Weifang, causing approximately 5%-15% economic loss across the survey area. In order to identify the pathogen, 17 isolates obtained from the diseased samples and the purified cultures exhibited the same morphological characteristics. Pathogenicity tests for the representative GM204 revealed that this fungus was the causal agent of the disease. Based on morphological characteristics, verification of Koch's postulates and homology analysis of the ITS and LSU sequences, the pathogen was identified as Mucor irregularis. To our knowledge, this is the first report of root rot on strawberry caused by M. irregularis in Shandong. Our findings will provide basic references for disease management of this pathogen.

由假禾谷镰刀菌(Fusarium pseudograminearum,Fp)引起的小麦茎基腐病是小麦生产上一种根茎部的真菌病害,严重影响小麦的产量和品质。为了开发防治小麦茎基腐病的链霉菌生防资源,本研究采用稀释涂布平板法和皿内对峙法从健康植株根际土壤中分离、筛选高效拮抗假禾谷镰刀菌的链霉菌。结果表明,10份供试土样中共分离到69株链霉菌,其中菌株YC2-3对假禾谷镰刀菌的拮抗作用最强,抑菌带宽度大于14 mm。基于系统发育分析、形态特征、培养特征和生理生化特性对YC2-3进行多相分类鉴定,将菌株YC2-3鉴定为直丝紫链霉菌(Streptomyces rectiviolaceus)。抑菌谱测试结果表明YC2-3具有广谱的抗真菌活性。温室盆栽试验结果表明,菌株YC2-3处理的治疗组和预防组均可以降低小麦茎基腐病的发病率和病情指数,生防效果分别为69.01%和73.94%。菌株YC2-3可以产生多种真菌细胞壁降解酶、铁载体和ACC脱氨酶,具有溶解有机磷的能力,还具有多种抗生素合成的核心基因。实时定量PCR(RT-qPCR)试验结果显示,YC2-3灌根处理12 h后,小麦茎基部组织中过氧化物酶基因TaPOD被激活表达。以上结果表明S. rectiviolaceus YC2-3具有开发成为小麦茎基腐病生防制剂的潜力。

类拟盘多毛孢真菌(Pestalotiopsis-like fungi)是危害蓝莓的一类重要病原菌。实验室前期对比发现有8株分离自蓝莓病样的类拟盘多毛孢菌单独聚成一支,并且与危害蓝莓的类拟盘多毛孢已知种相互区分,表明这8株菌有可能是危害蓝莓的新纪录种或新种。为明确这8株菌的种群分类地位,本研究对供试菌株进行形态学鉴定及系统发育分析,结果表明它们隶属于污斑新拟盘多毛孢Neopestalotiopsis foedans,这是该菌危害蓝莓在国内外的首次报道。在此基础上,进一步研究了该新纪录种不同菌株对辽宁省5个蓝莓主栽品种的致病能力,结果表明这8株菌的致病力具有明显分化,不同蓝莓品种均对强致病菌株较敏感,生产中应当加以重视。

稻瘟病菌引起的穗颈瘟严重影响水稻产量和质量。前期对江苏省稻区连续多年监测首次发现一类能引起类似穗颈瘟的梨孢菌新种Pyricularia sp. jiangsuensis,发生率高且在不同年份稳定存在。为了解该病原菌与稻瘟病菌之间的差异,本研究对该病原菌代表菌株P. sp. jiangsuensis 18-2与稻瘟病菌菌株P. oryzae Guy11在生长、产孢、各种胁迫及不同品种对两种病害的抗性等方面进行比较。结果表明,P. sp. jiangsuensis 18-2在PDA、CM和SDC三种培养基上的生长速度明显快于P. oryzae Guy11,但是P. sp. jiangsuensis 18-2的气生菌丝较为稀疏。在细胞壁胁迫因子和氧化压力下,P. sp. jiangsuensis 18-2对外源逆境的耐受性较P. oryzae Guy11更强,该病原菌更适应对外界胁迫的应答。进一步分析江苏省推广的主栽品种对类穗颈瘟的抗性,发现一些主栽品种对P. sp. jiangsuensis 18-2和P. oryzae Guy11引起的穗颈瘟存在抗性差异,鉴定筛选出品种镇稻448、盐粳15号、晶两优534和甬优2640对两者都表现良好的抗性,生产上应综合考虑选用对这两种病害抗性较好的品种。

Potato spindle tuber viroid (PSTVd) is a kind of viroid with great harm. In this study, high-throughput sequencing combined with sanger sequencing were used to identify possible viruses in Lycium Linn. samples, and PSTVd was identified in Lycium Linn. for the first time. Six pairs of primers were designed, and suitable primers were screened for RT-PCR detection of PSTVd. RT-PCR was carried out on 38 different varieties of Lycium Linn. samples, and the results showed that the detection rate for PSTVd was 52.6% (20/38). The complete genome sequences of 19 PSTVd isolates were obtained by cloning and sequencing. The sequence analysis showed that the nucleotide homology between the sequences was 96.1%-100%. The PSTVd Lycium Linn. isolates and the reported potato and tomato isolates were subjected to phylogenetic tree analysis, and the PSTVd isolates were divided into three groups, two of which only contained Lycium Linn. isolates. This study enriches the host range of PSTVd, and also provides a basis for future research on the pathogenicity of Lycium Linn. PSTVd.

为筛选对小麦茎基腐病安全且有效的药剂组合,本研究采用菌丝生长速率法检测不同作用机制的杀菌剂咯菌腈与戊唑醇复配对病原菌假禾谷镰孢的毒力,筛选出最佳增效组合,进而利用温室苗期生物测定明确复配药剂的安全性及防治效果,最后利用qPCR检测复配药剂对小麦根际土壤及茎秆中病原菌的影响。结果表明:咯菌腈与戊唑醇质量比为1∶7的复配药剂对假禾谷镰孢菌丝生长毒力最强,平均增效系数为7.22;该组合复配种衣剂在3个不同抗性小麦品种上温室苗期防治效果分别为73.35%、62.59%和82.07%,显著高于同剂量的两个单剂,且兼有促生作用;对小麦根长、鲜质量分别提高了2.81%～12.33%和18.87%～49.21%。通过qPCR检测发现,使用该复配药剂与空白对照相比假禾谷镰孢含量在小麦根际土壤中降低76.95%～90.37%,在小麦茎秆组织中降低92.00%～96.70%。验证了咯菌腈与戊唑醇质量比为1∶7的复配剂可通过降低土壤和小麦茎秆组织中病原菌的含量从而达到防治效果。本研究为小麦茎基腐病的化学防治提供新的参考依据。

III型分泌系统(Type Ⅲ secretion system, T3SS)是水稻细菌性条斑病菌(Xanthomonas oryzae pv. oryzicola, Xoc)的主要致病因子。实验室前期鉴定了一批十字花科黑腐病菌III型分泌系统的小分子抑制剂。这些抑制剂对Xoc的T3SS是否具有作用,值得探究。本研究利用Lux和GUS报告基因分别与T3SS相关基因启动子区融合构建Xoc融合报告系统,用报告系统以及定量PCR鉴定对Xoc T3SS有作用的小分子。结果表明,Carmofur、5-Fluorocytosine、A-3和Dealkaline lignin对Xoc T3SS基因表达具有显著抑制作用,A-3和Dealkaline lignin抑制Xoc生长,4种物质均未显著影响Xoc的致病力。为精确检测Xoc的过敏反应(hypersensitive response, HR)及小分子抑制剂的生防作用,本研究利用融合PCR及同源双交换原理构建能在辣椒ECW-10R上激发特异HR的报告菌株GX01/avrBs1。结果显示, GX01/avrBs1可在辣椒ECW-10R上快速激发HR,Dealkaline lignin可显著延迟Xoc在辣椒ECW-10R上的HR,进一步表明Dealkaline lignin抑制Xoc T3SS。

2020年河北省金银花主产区发生了一种金银花新病害,本文针对此病害开展了病原菌分离、鉴定及生物防治研究。采用常规组织分离法进行病原菌分离,通过盆栽试验完成分离物的致病性测定,通过形态学观察和分子生物学方法对分离物进行鉴定,并确定金银花病原菌的生理型、生理小种和交配型。同时在田间条件下评价微生物杀菌剂“10亿芽胞/克枯草芽胞杆菌可湿性粉剂”对金银花新病害的防治效果。从金银花病株维管束中分离获得7个真菌纯培养物,柯赫氏法则证明这7株真菌均为金银花新病害的病原菌。显微观察发现这7株真菌的分生孢子梗均具轮状分枝,在培养基上均可形成微菌核,据此初步确定其为轮枝菌属真菌(Verticillium)。rDNA-ITS序列比对和大丽轮枝菌特异性引物扩增证明这7株真菌均为大丽轮枝菌(V. dahliae)。进一步采用相关特异性引物扩增证明,这7株大丽轮枝菌均为非落叶型菌株、2号生理小种、MAT1-2-1交配型。致病性测定结果表明,金银花黄萎菌还能够侵染棉花、茄子、马铃薯、向日葵等作物,在4种作物上均表现出较强的致病性。田间采用微生物杀菌剂“10亿芽胞/克枯草芽孢杆菌可湿性粉剂”穴施可有效减轻金银花黄萎病的发生,防治效果达86.36%,增产11.7%。在我国首次报道由大丽轮枝菌引起的金银花黄萎病,“10亿芽胞/克枯草芽孢杆菌可湿性粉剂”可用于该病害的有效防控。

采集疑似朱顶红褪绿环斑病毒(HCRV)侵染的水鬼蕉(Hymenocallis littoralis)不同发病时期的叶部组织,通过HCRV-N基因特异性引物的RT-PCR扩增克隆与测序分析,确定由HCRV侵染。进一步应用负染色、超薄切片制样及透射电子显微镜观察,结果显示:叶部无症、褪绿、黄化和坏死不同发病时期的HCRV粒体在细胞内呈现散布或聚集的特征,散布的病毒粒体包被双层膜结构,发病后期聚集的病毒粒体呈现串珠状,聚集于管状囊泡中。在侵染潜伏期,水鬼蕉细胞核、叶绿体等亚细胞结构较完整;发病早期和中期,细胞核结构完整,叶绿体基粒片层消解,线粒体增加且结构较完整;发病后期,大部分细胞坏死,液泡中残存内溶物,少部分细胞的液泡中含有串珠状病毒粒体聚集于管状囊泡,是HCRV区别于同属其他病毒的明显特征,可作为诊断鉴定的依据。

During 2015-2016, Leaf samples of watermelon and melon exhibiting chlorotic and silver mottle symptoms were collected from Guangxi Zhuang Autonomous Region. Virus detection was done using double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and reverse transcription-polymerase chain reaction (RT-PCR). The results showed that 38 out of 143 samples were reacted positively against the antisera of watermelon silver mottle virus (WSMoV). Total RNA of the 38 positive samples were extracted and analyzed by RT-PCR using WSMoV specific primers. PCR amplicons with an expected size of 455 bp were obtained from all of the 38 samples, confirming that these samples were infected with WSMoV. Gene cloning, sequence comparison and phylogenetic trees construction were applied for the identification of the viruses. Two complete S RNA genome sequences were obtained (GenBank No.:OK905442 and OK905443), and the two sequences had 97% nucleotide identity with WSMoV-GZ (JX177645). Phylogenetic tree analysis showed that the WSMoV Guangxi watermelon isolates formed a small branch with WSMoV-GZ based on the nucleocapsid gene sequence. The results indicated that the WSMoV Guangxi watermelon isolates shared a close evolutionary relationship with WSMoV-GZ.

瓜类褪绿黄化病毒(cucurbit chlorotic yellows virus,CCYV)给我国葫芦科作物造成了严重的经济损失。2014—2019年持续调查山东葫芦科作物产区CCYV的发生情况。调查结果表明:CCYV可以自然侵染西瓜、甜瓜、南瓜、黄瓜和西葫芦,样品检出率由2014年的1.7%增加到2019年的29.1%,西葫芦地块发病严重有进一步蔓延的趋势。利用RT-PCR结合RACE技术扩增CCYV西葫芦分离物全基因组序列。结果表明:CCYV西葫芦分离株的基因组RNA1和RNA2与GenBank分离株的相似性均为99.6%~100%,基于CCYV全基因组的系统发育进化树均聚类在一个分支上,与各个蛋白的进化树和相似性分析结果一致,CCYV在传播的过程中与寄主无关但可能跟地理位置有关。本研究扩增分析了CCYV西葫芦分离物的基因组序列,对CCYV的变异现状和进化趋势研究具有一定的参考价值。

Anthracnose caused by Colletotrichum spp. is an important postharvest disease, which severely affects the quality of papaya fruits. In this study, fungal colonies were isolated and identified from diseased papaya fruits collected from Guangxi Province. Koch's rule was fulfilled by inoculating three fungal strains, MG2, MG15-4 and MG15-7 on papaya fruits, resulting in the similar symptoms of anthracnose. The strains were identified as C. truncatum based on morphological characteristics and molecular biological identification. This is the first report of C. truncatum caused anthracnose on papaya in China.

希金斯炭疽菌(Colletotrichum higginsianum)是一种重要的植物病原真菌,可引起严重的十字花科蔬菜炭疽病,对蔬菜生产造成了很大的影响。为了探究希金斯炭疽菌自噬相关基因ChAtg26在致病过程中的作用,本研究以希金斯炭疽菌侵染拟南芥Col-0后的cDNA为模板,通过qRT-PCR技术测定了ChAtg26基因在侵染过程中的表达模式,并利用同源重组技术构建了ChAtg26基因的敲除与回补突变体,分析了敲除ChAtg26基因对希金斯炭疽菌生长发育与致病能力的影响。结果表明,ChAtg26基因在病菌侵染寄主后0～40 h中有较高的表达量,而敲除ChAtg26基因后,病菌在生长速率、孢子萌发、附着胞形成以及对氧化胁迫敏感性方面没有明显变化,但是会在菌落黑色素累积与对细胞壁胁迫的敏感性方面有所下降,同时其产孢量与致病力会明显降低,说明ChAtg26基因参与了希金斯炭疽菌的黑色素合成、细胞壁胁迫应答反应、产孢与致病过程,并在这些过程中起着重要作用。

Black foot disease is one of the most important grapevine trunk diseases in the world. In this study, three samples from diseased grapevine trunks were collected from three vineyards in Beijing municipality and Hebei province. The potential pathogen was isolated, purified, and identified. In addition, pathogenicity of the isolates was tested to fulfill Koch′s postulates, and the effect of temperature on their mycelial growth was investigated. According to morphological characteristics and a multi-gene phylogenetic analysis with sequences of ITS, EF 1-α, HIS3, and TUB2, five isolates were identified as Dactylonectria novozelandica, which was the causal agent of grapevine black foot disease. Moreover, the pathogen was able to grow between 10 and 30 ℃. The optimum temperature for mycelial growth was 25 ℃. This is the first report of grapevine black foot disease caused by D. novozelandica in China.

To understand the pathogen causing leaf spot disease of date palm in Wenchang city, Hainanpro-vince. A fungal pathogen YZY47-3 was isolated from the diseased tissue and purified by single-spore isolation. According to morphological characteristics and combinatory phylogenetic analysis of ITS region, EF-1α and TUB gene sequences, the pathogen was identified as Neopestalotipsis clavispora. To the best of our knowledge, this is the first report of N. clavispora causing leaf spot disease of date palm in China.

In September 2021, a foliar anthracnose disease of kiwifruit was observed in Shankou base, Fengxin County, Jiangxi Province. Isolation were performed for the infected leaf tissues with the conventional tissue isolation method. On the basis of morphological characteristics, pathogenicity test and analysis of the combined sequence data of rDNA-ITS, TUB2 and CAL genes, the isolates were identified as Colletotrichum fructicola. To our knowledge, this is the first report of C. fructicola causing anthracnose of kiwifruit both in China and abroad.

在植物与病原菌的互作中,质外体效应子通过干扰植物的免疫系统,帮助病原菌成功定殖。大豆疫霉菌(Phytophthora sojae)分泌的质外体效应子XEG1是糖基水解酶12家族的蛋白,其能够被细胞膜上的模式识别受体RXEG1识别,诱导植物细胞坏死并触发免疫反应。本研究发现大豆疫霉菌分泌的XEG1同源蛋白Ps140300能够抑制XEG1诱导的细胞坏死和免疫反应。Ps140300是大豆疫霉菌的一个致病因子,其在大豆疫霉菌侵染早期上调表达。与XEG1不同,Ps140300不能被RXEG1识别诱导细胞坏死。免疫共沉淀实验显示Ps140300并不影响RXEG1与XEG1以及RXEG1与下游信号传递元件BAK1和SOBIR1的互作。进一步分析发现在不同病原菌中均存在可以抑制XEG1诱导细胞坏死的糖基水解酶12家族效应子。这些结果说明病原菌质外体效应子能够通过抑制寄主植物基础免疫,帮助其自身成功定殖。

条锈病是小麦上重要的真菌病害,严重威胁小麦的生产安全,施用杀菌剂是防治小麦条锈病的重要措施。三唑酮是防治条锈病的主要药剂,但长期单一、大规模使用三唑酮势必导致病原菌抗药性出现,由于小麦条锈菌是严格的专性寄生菌,目前测定条锈菌对杀菌剂的敏感性实验主要在活体小麦上进行,实验结果易受外界环境影响。本研究通过培养皿内离体小麦叶段培养,采取定量接种和软件读数的方法,对2021年西北核心越夏区60个小麦条锈菌菌株进行三唑酮敏感性测定,生测结果表明供试菌株平均EC₅₀值为0.543 μg·mL^-1,抗性菌株7株,敏感菌株53株,11.67%的供试菌株对三唑酮具有低至中等水平抗性,分子标记结果与生测结果高度吻合。本研究通过优化三唑酮对条锈病防治效果的室内生测体系,使得结果更加稳定、重复性好,同时基于敏感/抗药菌株靶标基因序列分析开发了高通量的抗药性竞争性等位基因特异性PCR-单核苷酸多态性分子标记(KASP-SNP),旨在为防治小麦条锈病筛选高效杀菌剂,同时开展田间小麦条锈菌抗药性监测,了解抗药性分布及抗性水平,为小麦条锈菌抗药性综合治理提供科学依据。

本研究采用菌丝生长速率法测定了中国不同地理来源的81株甜菜尾孢菌对多菌灵、苯醚甲环唑和氟硅唑的敏感性,并比较了5种杀菌剂的田间防效。结果显示,供试菌株中,对多菌灵敏感的菌株共57株,占总数的70.37%,抗性菌株24株,占总数的29.63%。苯醚甲环唑和氟硅唑对甜菜尾孢菌的平均EC₅₀值范围分别在0.003 5~3.818 0 μg·mL^-1和0.033 5~5.465 2 μg·mL^-1之间,平均EC₅₀值分别为(0.162 7±0.544 6)μg·mL^-1和(0.370 0±0.788 4)μg·mL^-1,最大EC₅₀值和最小EC₅₀值分别相差1 090倍和163倍。不同地区的菌株对苯醚甲环唑EC₅₀的最大值和最小值之比为2.3~853,部分地市的菌株对苯醚甲环唑敏感性差异显著,苯醚甲环唑平均EC₅₀值变化范围在0.031 0~1.915 9 μg·mL^-1。敏感性频率分布结果显示,甜菜尾孢菌对苯醚甲环唑已出现敏感性下降的亚群体,但82.7%的菌株敏感性频率呈正态分布,将此部分菌株的平均EC₅₀值(0.037±0.002)μg·mL^-1作为甜菜尾孢菌对苯醚甲环唑的敏感基线的参考值。不同地区的菌株对氟硅唑EC₅₀的最大值和最小值之比为1.3~65.6,部分地市的菌株对氟硅唑敏感性差异显著,氟硅唑平均EC₅₀值变化范围在0.131 2~1.093 7 μg·mL^-1。敏感性频率分布结果显示,甜菜尾孢菌对氟硅唑已出现敏感性下降的亚群体,但75.3%的菌株敏感性频率呈正态分布,将此部分菌株的平均EC₅₀值(0.145±0.007)μg·mL^-1作为甜菜尾孢菌对氟硅唑的敏感基线的参考值。抗性频率分析显示,甜菜尾孢菌对苯醚甲环唑及氟硅唑的抗性频率分别为20.99%和16.05%,抗性水平以低抗和高抗为主。田间药效试验结果表明,40%氟硅唑EC(40% Flusilazole EC)有效成分24 g·hm^-2、70%甲基硫菌灵WP(70% Thiophanate-methyl WP)有效成分630 g·hm^-2连续两次施药,距末次施药后10 d对甜菜褐斑病的田间防效达到65%左右,显著高于70%代森锰锌 WP(70% Mancozeb WP)有效成分1 050 g·hm^-2和50%异菌脲WP(50% Iprodione WP)600 g·hm^-2的防效,与10%苯醚甲环唑WG(10% Difenoconazole WG)52.5 g·hm^-2防效无显著差异。本研究结果为杀菌剂的合理使用及甜菜褐斑病的有效防治提供数据支持。

稻叶黑粉病是由担子菌Entyloma oryzae侵染水稻叶片或叶鞘引起的真菌病害,但对病原菌与寄主互作的细胞学机制一直缺少了解。本文对采自田间自然发病叶片上的病斑进行了初步的细胞学分析,结果发现,病原菌侵染后,寄主病斑部位的表皮细胞外部形态基本保持完整;病原菌主要在寄主叶肉细胞部位产生大量的厚垣孢子并逐渐取代叶肉细胞;病原菌菌丝在寄主胞外扩展,未见其穿透寄主细胞壁进入细胞内,也没有产生典型的真菌吸器。靠近病原菌菌丝的寄主各种细胞内的细胞器均发生降解,降解产生的脂类物质凝聚成了体积较大的脂质球。寄主维管束组织的细胞壁一直保持完整,未发现病原菌菌丝进入维管束,病原菌菌丝和孢子被限制在寄主相邻两个维管束之间。在发病后期,由于寄主叶片表皮结构整体性破坏导致大量细菌进入,加速了叶片的衰老死亡。本研究结果表明,稻叶黑粉病菌的侵染模式为胞外侵染,类似于活体营养真菌;但病原菌的侵染导致附近寄主细胞降解死亡,类似于腐生营养真菌。

浙江省金华市和丽水市出现了会导致茭白叶鞘产生褐色斑点的病害,感染该病害的植株会产生短而发黄的茭白。采用组织分离法从病样中分离得到XYQ3菌株。通过形态学观察、多序列比对分析和致病性试验,确定茭白叶鞘病的病原菌为Microdochium seminicola。在实验室条件下,对田间常用的12种杀菌剂进行毒性试验,结果表明,250 g·L^-1咪鲜胺乳油对病原菌菌丝体的抑制效果最好,EC₅₀值为0.067 mg·L^-1;其次是300 g·L^-1苯并丙环唑乳油和60%苯醚甲环唑WG,EC₅₀值分别为0.373和1.083 mg·L^-1。综合考虑,250 g·L^-1咪鲜胺乳油为最佳杀菌剂,50%异菌脲可湿性粉剂可作为替代品。本研究为茭白叶鞘褐斑病的鉴定和防治提供了科学依据。

From 2019 to 2021, grape white rot was reported in Shandong Province. In this study, we performed the isolation from the diseased samples from five different regions, and fifty fungal strains were obtained. The further analyses for 25 strains out of 50 based on morphological observation and multi-locus sequence analyses of ITS, EF-1α, and LSU were conducted and all identified as Coniella vitis. The pathogenicity tests on detached grape leaves, fruits, or living plants for five representatives from the 25 strains exhibited the typical symptoms of the disease , which were similar with that in the field conditions. To the best of our knowledge, it was the first time that C. vitis was identified to be the pathogen of grape white rot disease in Shandong Province, which will provide a scientific basis for the diagnosis and control.

谷子白发病是由病原卵菌禾生指梗霉(Sclerospora graminicola)引起的一种严重影响谷子生产的土传病害。在谷穗生长锥伸长期,禾生指梗霉侵染扩展至谷子穗部可导致谷穗分化发育受阻,形成畸形穗。为解析禾生指梗霉侵染影响谷子幼穗分化及发育的分子基础,本研究观测了受侵染谷穗在生长锥突起期(S1)至雌雄蕊分化期(S5)的形态变化,借助RNA-seq分析谷子穗部响应禾生指梗霉侵染的差异表达基因及关键调控因素,并通过LC-MS/MS测定病穗中生长素的含量变化。研究结果表明,受侵染谷穗在生长锥突起期发育受阻,枝梗分化期无明显一级、二级指梗分化特征;共有336个基因在谷穗发育不同时期(S1~S5)均差异表达,显著富集在激素介导的信号通路、激素水平调节、激素代谢过程、激素生物合成过程等GO通路,通过KEGG也富集到色氨酸代谢、类胡萝卜生物合成、玉米素生物合成等与激素生物合成相关的途径,其中在色氨酸代谢通路富集到328个基因;IAA生物合成通路与转运通路相关调控基因表达量上调,而在IAA信号转导通路相关基因表达量下降。在S4时期病穗中生长素含量显著高于对照组,IAA处理可促进菌丝的生长发育。研究结果表明:禾生指梗霉可能通过调控寄主体内生长素的合成促进病原菌向谷子生长点进一步侵染和扩展。

Yam latent virus is an important virus that infects yam. It can cause mosaics and chlorosis on yam leaves and affect both the quality and yield. In this study, 6 specific primer pairs were designed according to the cp gene sequence of yam latent virus (YLV), and a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay with strong specificity and high sensitivity was established. The target product was amplified at 50-65 ℃ for 1 hour, and the optimal reaction temperature was 58-65 ℃. The specificity result showed that this method was able to specifically identify YLV from the other three RNA viruses infecting yam. The sensitivity test showed that the detection limit of the LAMP method was 5.08×10¹ copies·μL^-1. The sensitivity of this method was 100 times that of the conventional PCR. The results indicated that this method can accurately detect YLV from the field samples. The RT-LAMP established in this study is sensitive, specific and reliable, thus making it suitable for the rapid detection of YLV.