Antagonistic Potential of Bacterial Species against Fungal Plant Pathogens(FPP)and Their Role in Plant Growth Promotion(PGP):A Review

Since the 19th century to date,the fungal pathogens have been involved in causing devastating diseases in plants.All types of fungal pathogens have been observed in important agricultural crops that lead to significant pre and postharvest losses.The application of synthetic fungicide against the fungal plant pathogens(FPP)is a traditional management practice but at the same time these fungicides kill other beneficial microbes,insects,animal,and humans and are harmful to environment.The antagonistic microorganism such as bacteria are being used as an alternate strategy to control the FPP.These antagonistic species are cost-effective and eco-friendly in nature.These biocontrol bacteria have a broad mechanism against fungal pathogens present in the phyllosphere and rhizosphere of the plant.The antagonistic bacteria have different strategies against the FPP,by producing siderophore,biofilm,volatile organic compounds(VOCs),through parasitism,antibiosis,competition for limited resources and induce systemic resistance(ISR)in the host plant by activating the immune systems.The commercial bio-products synthesized by the major bacterial species Pseudomonas syringae,Burkholderia cepacia,Streptomyces griseoviridis,Pseudomonas fluorescens and Bacillus subtilis are used to control Fusarium,Pythium,Rhizoctonia,Penicillium,Alternaria,and Geotrichum.The commercial bio-formulations of bacteria act as both antifungal and plant growth regulators.The Plant growth-promoting rhizobacteria(PGPR)played a significant role in improving plant health by nitrogen-fixing,phosphorus solubilization,phytohormones production,minimizing soil metal contamination,and by ACC deaminase antifungal activities.Different articles are available on the specific antifungal activity of bacteria in plant diseases.Therefore,this review article has summarized the information on biocontrol activity of bacteria against the FPP and the role of PGPR in plant growth promotion.This review also provided a complete picture of scattered information regarding antifungal activities of bacteria and the role of PGPR.

In Vitro Antifungal Activity of the Extract and Compound from Acorus tatarinowii Against Seven Plant Pathogenic Fungi

Acorus tatarinowii Schott is a traditional Chinese medicine plant and has multiple bioactivities in medicine and pesticide field.In this study,the antifungal compound 1,2-dimethoxy-4(2-propenyl) benzene was isolated from A.tatarinowii Schott by activity-directed isolation method,and the inhibitory activity of the extract and 1,2-dimethoxy-4(2-propenyl) benzene against seven plant pathogenic fungi was evaluated.The results showed that the extract and 1,2-dimethoxy-4(2propenyl) benzene had high inhibitory activity against hyphal growth of Thielaviopsis paradoxa(de Seynes) V.Hohnel,Pestalotia mangiferae P.Henn.,Fusarium oxysporum f.sp.niveum(E.F.Smith) Syn.et Hans.,Alternaria alternate Tanaka,Colletotrichum musae(Berk et Curt) V.Arx,Sphaceloma fawcettii Jenk.,and Mycosphaerella sentina(Fr.) Schroter.The EC50 values of extract were 1.6162,1.6811,1.1253,3.5771,1.7024,2.2284,and 2.2221 g L-1,respectively,and the EC50 values of 1,2-dimethoxy-4(2-propenyl) benzene were 0.1021,0.0997,0.0805,0.1742,0.1503,0.1853,and 0.1924 g L-1,respectively.1,2-Dimethoxy-4(2-propenyl) benzene also inhibited spores germination of T.paradoxa(de Seynes) V.Hohnel and F.oxysporum f.sp.niveum(E.F.Smith) Syn.et Hans.,with the inhibitory rates of 98.81 and 100% at a concentration of 0.4 g L-1 after 8 h,respectively.1,2-Dimethoxy-4(2-propenyl) benzene is a potential botanical antifungal agent for controling of plant fungal diseases.

Functional identification of phenazine biosynthesis genes in plant pathogenic bacteria Pseudomonas syringae pv. tomato and Xanthomonas oryzae pv. oryzae

Phenazines are secondary metabolites with broad spectrum antibiotic activity and thus show high potential in biological control of pathogens.In this study,we identified phenazine biosynthesis(phz)genes in two genome-completed plant pathogenic bacteria Pseudomonas syringae pv.tomato(Pst)DC3000 and Xanthomonas oryzae pv.oryzae(Xoo)PXO99~A.Unlike the phz genes in typical phenazine-producing pseudomonads,phz homologs in Pst DC3000 and Xoo PXO99~A consisted of phzC/D/E/F/G and phzC/E1/E2/F/G,respectively,and the both were not organized into an operon.Detection experiments demonstrated that phenazine-1-carboxylic acid(PCA)of Pst DC3000 accumulated to 13.4μg L^(-1),while that of Xoo PXO99~A was almost undetectable.Moreover,Pst DC3000 was resistant to 1 mg mL^(-1)PCA,while Xoo PXO99~A was sensitive to50μg mL^(-1)PCA.Furthermore,mutation of phzF blocked the PCA production and significantly reduced the pathogenicity of Pst DC3000 in tomato,while the complementary strains restored these phenotypes.These results revealed that Pst DC3000 produces low level of and is resistant to phenazines and thus is unable to be biologically controlled by phenazines.Additionally,phz-mediated PCA production is required for full pathogenicity of Pst DC3000.To our knowledge,this is the first report of PCA production and its function in pathogenicity of a plant pathogenic P.syringae strain.

Effectiveness of Ringworm Cassia and Turmeric Plant Extracts on Growth Inhibition against Some Important Plant Pathogenic Fungi

Crude plant extracts of ringworm cassia, Cassia alata L. and turmeric, Curcuma longa L. were prepared by either hot water or organic solvents such as ethanol and ether. Various concentrations of the crude extract were then subjected to an in vitro test for their effectiveness on mycelia growth inhibition against some important plant pathogenic fungi such as Alternaria alternata, Colletotrichum gloeosporioides, Fusarium oxysporum fsp. lycopersici, Sclerotium rolfsii, Phytophthora infestans and Pythium sp. in comparison to commercial fungicides such as copper oxychloride and mancozeb. Reduction of the fungal growth was significantly obtained with C. longa extracts and the best median effective inhibitory concentration (IC50) value of 6.07, 6.50 and 7.13 mg/ml was from the ethanol extract for S. rolfsii, C. gloeosporioides and F. oxysporum fsp. lycopersici respectively. While all extracts from C. alata were almost the least effective against these fungi. The efficacy of C. longa extracts therefore, provided an alternative regime for the control of the fungal diseases and a promising appreciable choice for a replacement of chemical fungicides.

Phytochemical Determination and Antibacterial Activity of Punica granatum Peel Extracts against Plant Pathogenic Bacteria

Plant pathogenic bacteria are recognized to be harmful microbes able to decrease the quantity and quality of crop production in the world. Punica granatum peel was screened for its potential use as biological control agent for plant pathogenic bacteria. P. granatum peel was successfully extract using n-hexane, methanol and ethyl acetate by maceration. The highest yield obtained by ethyl acetate showed that ethyl acetate extracted more compounds that readily soluble to methanol and n-hexane. For in-vitro antibacterial activity, three different species of plant pathogenic bacteria were used namely Erwinia carotovorum subsp. Carotovorum, Ralstonia solanacearum, and Xanthomonas gardneri. For all crude extracts, four different concentrations 25, 50, 100 and 200 mg/ml were used in cup-plate agar diffusion method. Streptomycin sulfate at concentration 30 μg/ml was used as positive control while each respective solvent used for peel extraction was used as negative control. The results obtained from in vitro studies showed only ethyl acetate extract possessed antibacterial activity tested on the plant pathogenic bacteria. Methanol and n-hexane did not show any antibacterial activity against plant pathogenic bacteria selected where no inhibition zones were recorded. R. solanacearum recorded the highest diameter of inhibition zones for all range of concentrations introduced followed by E. carotovorum subsp. Carotovorum and X. gardneri. For the minimum inhbitory concentration (MIC) and minimum bactericidal concentration (MBC), only the ethyl acetate extract was subjected to the assay as only ethyl acetate extract exhibited antibacterial activity. The minimum concentration of ethyl acetate extract that was able to inhibit plant pathogenic bacteria was recorded at a concentration of 3.12 mg/ml which inhibited R. solancearum and E. carotovorum subsp. Carotovorum, followed by X. gardneri at concentration 6.25 mg/ml. For the minimum bactericidal concentration (MBC), the results showed that at the concentration of 12.5 mg/ml, the extract was still capable of killing the pathogenic bacteria, R. solanacearum, and P. caratovora sub.sp. caratovora while for the bacteria X. gardneri, the concentration that was able to kill the bacteria was 25 mg/ml. The qualitative estimation of phytochemical constituents within P. granatum L. ethyl acetate peel extracts had revealed the presence of tannins, flavonoids, phenols alkaloid, Saponins, and terpenoids. This study has demonstrated that Ethyl Acetate peel extracts of P. granatum has significant antibacterial activity against pathogenic plant bacterial, and it could be of high agricultural value.

IN VITRO ACTIVITY OF EXTRACTS OF FIVE MEDICINAL PLANT SPECIES ON PLANT PATHOGENIC FUNGI

The antifungal effectiveness of extracts of five medicinal plant species was determined.The inhibitory activity of extracts of Eucalyptus tereticornis,Xanthium sibiricum,Artemisia argyi,Tupistra chinensis and Pyrola calliantha were evaluated against the mycelial growth of the plant pathogenic fungi Aspergillus niger,Botrytis cinerea,Penicillium digitatum,P.expansum,P.italicum and Rhizopus stolonifer.All plant extracts were prepared at 60°C using solvents(either water,50%ethanol(v/v),95%ethanol(v/v),ethyl acetate or petroleum ether).Fungicidal effects of all plants tested were confirmed.Different extracts from the same plant species gave different degrees of inhibition.All aqueous extracts had weak or no activity on all fungi tested.Ethyl acetate and 95%ethanol extracts from T.chinensis rhizomes gave greater inhibition and a broader spectrum inhibition than the other extracts.T.chinensis may have potential as a new natural fungicide and may be used for the preservation of agricultural and forestry products such as fruits and vegetables.

Pathogenesis-related protein genes involved in race-specific allstage resistance and non-race specific high-temperature adultplant resistance to Puccinia striiformis f. sp. tritici in wheat

Interactions of the stripe rust pathogen(Puccinia striiformis f. sp. tritici) with wheat plants activate a wide range of host responses. Among various genes involved in the plant-pathogen interactions, the expressions of particular pathogenesisrelated(PR) protein genes determine different defense responses. Different types of resistance have been recognized and utilized for developing wheat cultivars for resistance to stripe rust. All-stage resistance can be detected in seedling stage and remains at high levels throughout the plant growth stages. This type of resistance is race-specific and not durable. In contrast, plants with only high-temperature adult-plant(HTAP) resistance are susceptible in seedling stage, but become resistant when plants grow older and the weather becomes warmer. HTAP resistance controlled by a single gene is partial, but usually non-race specific and durable. The objective of this study was to analyze the expression of PR protein genes involved in different types of wheat resistance to stripe rust. The expression levels of 8 PR protein genes(PR1, PR1.2, PR2, PR3, PR4, PR5, PR9 and PR10) were quantitatively evaluated at 0, 1, 2, 7 and 14 days after inoculation in single resistance gene lines of wheat with all-stage resistance genes YrTr1, Yr76, YrSP and YrExp2 and lines carrying HTAP resistance genes Yr52, Yr59, Yr62 and Yr7 B. Races PSTv-4 and PSTv-37 for compatible and incompatible interactions were used in evaluation of PR protein gene expression in wheat lines carrying all-stage resistance genes in the seedlingstage experiment while PSTv-37 was used in the HTAP experiment. Analysis of quantitative real-time polymerase chain reaction(qRT-PCR) revealed that all of the PR protein genes were involved in the different types of resistance controlled by different Yr genes. However, these genes were upregulated at different time points and at different levels during the infection process among the wheat lines with different Yr genes for either all-stage resistance or HTAP resistance. Some of the genes were also induced in compatible interactions, but the levels were almost always higher in the incompatible interaction than in the compatible interaction at the same time point for each Yr gene. These results indicate that bothsalicylic acid and jasmonate signaling pathways are involved in both race-specific all-stage resistance and non-race specific HTAP resistance. Although expressing at different stages of infection and at different levels, these PR protein genes work in concert for contribution to different types of resistance controlled by different Yr genes.

Proteomics: A Successful Approach to Understand the Molecular Mechanism of Plant-Pathogen Interaction

In recent years, proteomics has played a key role in identifying changes in protein levels in plant hosts upon infection by pathogenic organisms and in characterizing cellular and extracellular virulence and pathogenicity factors produced by pathogens. Proteomics offers a constantly evolving set of novel techniques to study all aspects of protein structure and function. Proteomics aims to find out the identity and amount of each and every protein present in a cell and actual function mediating specific cellular processes. Structural proteomics elucidates the development and application of experimental approaches to define the primary, secondary and tertiary structures of proteins, while functional proteomics refers to the development and application of global (proteome wide or system-wide) experimental approaches to assess protein function. A detail understanding of plant defense response using successful combination of proteomic techniques and other high throughput techniques of cell biology, biochemistry as well as genomics is needed for practical application to secure and stabilize yield of many crop plants. This review starts with a brief introduction to gel- and non gel-based proteomic techniques followed by the basics of plant-pathogen interaction, the use of proteomics in recent pasts to decipher the mysteries of plant-pathogen interaction, and ends with the future prospects of this technology.

Identification of Plant-Pathogenic Fungi Using Fourier Transform Infrared Spectroscopy Combined with Chemometric Analyses

Identification of plant-pathogenic fungi is time-consuming due to cultivation and microscopic examination and can be influenced by the interpretation of the micro-morphological characters observed.The present investigation aimed to create a simple but sophisticated method for the identification of plant-pathogenic fungi by Fourier transform infrared(FTIR)spectroscopy.In this study,FTIR-attenuated total reflectance(ATR)spectroscopy was used in combination with chemometric analysis for identification of important pathogenic fungi of horticultural plants.Mixtures of mycelia and spores from 27fungal strains belonging to nine different families were collected from liquid PD or solid PDA media cultures and subjected to FTIR-ATR spectroscopy measurements.The FTIR-ATR spectra ranging from 4 000to 400cm-1 were obtained.To classify the FTIRATR spectra,cluster analysis was compared with canonical vitiate analysis(CVA)in the spectral regions of3 050~2 800and 1 800~900cm-1.Results showed that the identification accuracies achieved 97.53%and99.18%for the cluster analysis and CVA analysis,respectively,demonstrating the high potential of this technique for fungal strain identification.

Antimicrobial Activity of Wild Plant Seed Extracts against Human Bacterial and Plant Fungal Pathogens

Five wild plant species belonging to different families (Chenopodium album, Plantago major, Elytrigia elongata, Filipendula ulmaria and Nigella sativa) widely spread in Russian Federation and the former USSR were evaluated for their ability to inhibit growth of two important human food-borne pathogens (Escherichia coli O157:H7 and Listeria monocytogenes strain EGD-e) and eight plant pathogens (Alternaria alternata, Alternaria tenuissima, Bipolaris sorokiniana, Stagonospora nodorum, Fusarium solani, Fusarium oxysporum, Fusarium culmorum and Phytophtora infestans). To isolate biologically active compounds from seeds, a step-wise procedure including extraction with hexane, ethyl acetate, ethanol, and 10% acetic acid followed by reversed-phase HPLC was developed. Using disc-diffusion assay, the highest activity against E. coli O157:H7 was observed with extracts from F. ulmaria (hexane and ethyl acetate extracts and the unbound RP-HPLC fraction) and P. major (ethyl acetate extract and the unbound RP-HPLC fraction);E. elongate (the unbound RP-HPLC fraction) was less active. The extracts from P. major and E. elongate (the unbound RP-HPLC fractions) were equally highly active against L. monocytogenes, while those of F. ulmaria (the unbound RP-HPLC fraction) and N. sativa (hexane and ethyl acetate extracts) were less active against this pathogen. The dynamics of L. monocytogenes EGD-е and E. coli O157:H7 growth in the presence of two most potent extracts (RP-HPLC-unbound fractions of P. major and E. elongate and the hexane extract of F. ulmaria) was studied.

Correlation between Chemical Structure and Biological Activity of Host-Selective Plant Pathogen Mycotoxins

Toxin, one of the most important factors of plant fungal disease, has attracted much attention of many academicians who have been studying pathogen mycotoxin in deep research. The paper summarized chemical structures of some host-selective plant pathogen mycotoxins discovered in recent years and the correlation between biological activity and chemical structure of toxin.

茶皂素和博落回生物碱复配对4种病原菌的联合毒力

为了明确博落回生物碱与茶皂素混配对棉花立枯病菌、棉花枯萎病菌、苹果轮纹病菌和柑橘炭疽病菌的联合毒力,采用生长速率法测定了博落回生物碱、茶皂素及其不同配比混剂对4种病菌的毒力,通过交互测定法测定了茶皂素和博落回生物碱的毒性比例和增效系数。结果表明:博落回生物碱与茶皂素复配对棉花枯萎病的联合毒力主要为拮抗或相加作用,博落回生物碱与茶皂素复配对苹果轮纹病的联合毒力主要为相加作用,博落回生物碱与茶皂素质量比为1∶48.7的混剂对棉花立枯病表现为增效作用,增效系数为1.536;博落回生物碱与茶皂素质量比为1∶20.2的混剂对柑橘炭疽病菌表现出较好的增效作用,增效系数为1.537。

鱼腥草乙酸乙酯萃取部位的化学成分及抗植物病原菌活性研究

采用硅胶、大孔树脂、葡聚糖凝胶、MCI树脂、薄层色谱以及高效液相色谱等色谱分离方法,从鱼腥草(Houttuynia cordata)地上部分95%乙醇提取物的乙酸乙酯萃取部位分离得到10个化合物。运用质谱、核磁共振等波谱技术和文献对比法鉴定了其结构,分别鉴定为去氢催吐萝芙木醇(1)、5,6-环氧-3-羟基-β-紫罗兰酮(2)、黑麦草内酯(3)、pubinernoid A(4)、槲皮苷(5)、槲皮素(6)、阿福豆苷(7)、4-甲氧基苯-1,2-二醇(8)、4-羟基苯甲醛(9)、苯甲酸(10)。化合物1~4为首次从鱼腥草中分离得到的降碳倍半萜。对所有化合物进行了抗植物病原菌活性筛选,结果显示:在40 mg/mL浓度下,化合物5~7对禾谷镰刀菌、褐枝孢菌、番茄灰霉病菌、瓜类球腔菌和棉花枯萎病菌抑菌率介于38%~50%之间。

我国作物病菌标准品的研究进展

作物在生长发育过程中,容易受到病菌侵染,造成巨大的经济损失。及时准确地对作物感染病菌的情况进行检测,有助于为制定病害的防治措施提供参考依据。作物病菌标准品是作物病害检测工作中的重要参考,在作物病菌的定性和定量检测工作中具有十分重要的地位。目前,作物病菌标准品相关研究的报道较少,因此有序开展作物病菌标准品的研制工作极为迫切。本文通过对作物病菌检测方法、检测标准及作物病菌标准品研究进展进行梳理总结,以期为我国作物病菌标准品的研制提供理论参考。

独蒜兰提取物对植物病原真菌的抑菌活性

【目的】研究独蒜兰假鳞茎乙醇提取物对植物病原真菌的抑菌作用,为植物源杀菌剂的开发提供依据。【方法】采用菌丝生长速率法,研究独蒜兰假鳞茎乙醇提取物对15种植物病原菌的抑制活性;以西瓜尖孢镰刀菌作为供试菌,进一步研究该提取物对病原真菌的菌丝干重、细胞膜、过氧化氢酶(CAT)、过氧化物酶(POD)和超氧化物歧化酶(SOD)等的影响。【结果】独蒜兰假鳞茎提取物对辣椒疫霉病菌、西瓜尖孢镰刀菌、番茄灰霉病菌和非洲隐地疫霉菌的抑菌效果明显,其EC_(50)值分别为0.849、0.782、0.813和1.161 mg·mL^(-1);经独蒜兰提取物处理后的西瓜尖孢镰刀菌菌丝干重随着药剂浓度的增加而减少;细胞膜丙二醛含量和相对电导率增加;菌丝体细胞内CAT、POD和SOD 3种保护酶活性增加。【结论】独蒜兰假鳞茎提取物对植物病原真菌具有较好的抑菌活性,其抑菌作用可能与其干扰菌丝生长、使菌丝细胞膜正常功能受损等有关。

镰刀菌属真菌毒素在植物和病原菌互作中的研究进展

镰刀菌是世界上最重要的植物病原菌之一,可影响植物的生长发育,严重威胁全球粮食安全和生物多样性。几乎所有的镰刀菌都会产生真菌毒素,其毒素种类多、毒性强,一方面可以作为致病因子之一参与镰刀菌的致病过程,另一方面可污染粮食和饲料,进而引起人类和动物的相关病症。已有研究表明,镰刀菌侵染植物后产生的不同种类真菌毒素不仅毒害植物细胞,引起植物组织的坏死,还会加速病原菌的侵染;同时,针对病原菌产生的毒素,植物会激活防御酶并启动防御相关基因的表达,或将致病毒素转化为无毒或低毒物质并转运到胞外,或通过分泌次生代谢物直接抑制病原菌毒素的生物合成。为全面解析镰刀菌毒素在病原菌侵染植物中的作用,提高植物对病原菌的抗性,该文综述了镰刀菌属真菌毒素的种类、毒性机理以及毒素在植物和病原菌互作中的作用,并讨论了植物对真菌毒素的防御反应策略,以期为镰刀菌毒素致病机制和病原菌防治策略研究提供参考。

A LAMP-assay-based specific microbiota analysis reveals community dynamics and potential interactions of 13 major soybean root pathogens

Soybean root diseases are associated with numerous fungal and oomycete pathogens;however,the community dynamics and interactions of these pathogens are largely unknown.We performed 13 loop-mediated isothermal amplification(LAMP)assays that targeted specific soybean root pathogens,and traditional isolation assays.A total of 159 samples were collected from three locations in the Huang-Huai-Hai region of China at three soybean growth stages(30,60,and 90 days after planting)in 2016.In LAMP results,we found that pathogen communities differed slightly among locations,but changed dramatically between soybean growth stages.Phytophthora sojae,Rhizoctonia solani,and Fusarium oxysporum were most frequently detected at the early stage,whereas Phomopsis longicolla,Fusarium equiseti,and Fusarium virguliforme were most common in the later stages.Most samples(86%)contained two to six pathogen species.Interestingly,the less detectable species tended to exist in the samples containing more detected species,and some pathogens preferentially co-occurred in diseased tissue,including P.sojae–R.solani–F.oxysporum and F.virguliforme–Calonectria ilicicola,implying potential interactions during infection.The LAMP detection results were confirmed by traditional isolation methods.The isolated strains exhibited different virulence to soybean,further implying a beneficial interaction among some pathogens.

无子刺梨内生真菌拮抗活性菌株筛选及分子鉴定

为进一步开发利用无子刺梨枝条和叶片中具有生物防治潜力的内生真菌,以2种木腐菌和4种植物病原真菌为靶标,采用五点对峙法筛选对病原真菌具有拮抗活性的内生真菌菌株,并探讨活性菌株的分类地位。结果显示,菌株RSZF 36、RSZF 39和RSYF 34对2种木腐菌的抑制率均达80%以上,菌株RSZF 34和RSZF 36对4种植物病原真菌的抑制率均大于75%,菌株RSZF 36对6种病原菌的抑制率均高于75%,呈现强抑制能力。84株内生真菌中有12株对6种病原菌抑制率均在70%以上,属于活性菌株,其中9株来自枝条,3株来自叶片。对12株活性菌株进行分子生物学鉴定,将其归属为1门2纲3目3科4属,拟盘多毛孢属(Pestalotiopsis)为优势菌属,占活性菌株的41.67%。综上,无子刺梨内生真菌中广泛分布着抑菌活性菌株,且不同内生菌株对不同病原菌的拮抗能力表现出一定的选择性或专一性。本研究结果可为无子刺梨内生真菌资源的开发利用提供理论参考。

福建省茶树病毒种类鉴定及多重PCR检测技术的建立

【目的】明确福建省茶树主要病毒种类和分布情况,并建立同时快速检测多种病毒的多重PCR检测技术。【方法】2019—2023年,从福建省福州、南平、宁德、泉州、漳州、厦门、三明、莆田和龙岩9个地市区采集具有褪绿、皱缩和坏死等疑似病毒感染症状的茶树样品1869份,采用高通量测序技术结合PCR和RT-PCR检测的方法对茶树病毒病的病原进行鉴定,并将PCR扩增获得的特异性目的片段进行克隆和测序,对获得的序列进行系统发育分析;同时,根据GenBank上已报道的病毒序列设计特异性引物,通过退火温度、引物浓度、循环数等反应条件和反应程序的优化,建立同时检测福建茶树主要病毒的多重PCR检测技术,并测定该技术的特异性、灵敏度及实际应用效果。【结果】从所采集的茶树病样上检出3种病毒,按检出率从高到低依次为油茶双生病毒(oil tea associated geminivirus,OTaGV)(48.90%)、茶树坏死环斑病毒(tea plant necrotic ring blotch virus,TPNRBV)(26.75%)和茶树潜隐病毒1(camellia cryptic virus 1,CCV1)(17.98%);在检出病毒的1258份样品中,有807份为OTaGV、TPNRBV或CCV1单独侵染,检出率分别为37.20%、21.38%和5.56%;其余451份样品为2种或3种病毒复合侵染,复合侵染检出率达35.85%,OTaGV+CCV1、TPNRBV+CCV1、TPNRBV+OTaGV、OTaGV+CCV1+TPNRBV 4种类型复合侵染检出率分别为17.49%、0.40%、14.71%、3.26%。在地理分布上,OTaGV在福州、南平、宁德、泉州、漳州、厦门、三明、莆田和龙岩9个地区均有分布,其中漳州OTaGV检出率最高,为96.77%;CCV1在福州、南平、宁德、泉州、漳州、三明、莆田和龙岩8个地区均有分布,其中三明CCV1检出率最高,为66.00%;TPNRBV在福州、南平、宁德、泉州和漳州5个地区均有分布,其中泉州TPNRBV检出率最高,为79.13%;在福建省9个地区中,厦门地区仅检出OTaGV,三明、莆田和龙岩地区检出OTaGV和CCV1,其他5个地区同时检出OTaGV、TPNRBV和CCV1;福州、南平、宁德、泉州、漳州、三明、莆田7个地区检测到病毒复合侵染,其中漳州地区病毒复合侵染检出率最高(85.00%)、宁德地区病毒复合侵染检出率最低(23.03%)。利用测定的CCV1和TPNRBV部分基因序列构建系统发育树,结果表明本研究获得的CCV1分离物(FW)与已报道的福建分离物FJ-SH104(GenBank登录号:ON807095)亲缘关系最近、TPNRBV分离物(FU)与福建分离物QZHA92(GenBank登录号:OQ948454)亲缘关系最近。经优化建立的多重PCR检测技术特异性强,仅OTaGV、TPNRBV和CCV1能扩增出特异性目的片段,而其他病毒及健康茶树样品上均未扩增出特异性目的片段;多重PCR灵敏度最低可以检测到稀释至10^(-4)倍的OTaGV、TPNRBV和10^(-3)倍的CCV1;利用该多重PCR检测技术对茶园中采集的60份病样进行检测,检测结果与单一PCR检测结果完全相符。【结论】OTaGV、TPNRBV和CCV1是当前福建茶树上主要病毒种类,其中OTaGV为福建地区首次报道,该病毒可侵染茶树也为首次发现;在检出的3种病毒中,以OTaGV发生分布范围最广,其次为CCV1、TPNRBV;福建地区茶树病毒目前仍以单独侵染为主,但同时存在较多的复合侵染,复合侵染类型包括TPNRBV+CCV1、TPNRBV+OTaGV、CCV1+OTaGV和OTaGV+CCV1+TPNRBV;建立的多重PCR检测技术特异性强、灵敏度高,可用于茶园茶树上OTaGV、CCV1和TPNRBV3种病毒的快速检测。研究结果可为福建茶树病毒病的综合防控提供理论依据和技术支持。