Characterization of Erwinia sp. causing black rot of papaya (Carica papaya) first recorded in Okinawa Main Island,Japan

Since 2002, papaya black rot has been spreading over several islands of Okinawa Prefecture, Japan. To devise a prevention strategy for the disease, microbiological research on the pathogen was conducted. Twelve strains were isolated from papaya infected with black rot showing symptoms such as water-soaked lesions on stems and petioles, black spots on fruits, and rotted leaves turning yellow with necrotic spots. Through Koch's postulates, we confirmed that the isolated strains caused papaya black rot. Bacteriological assays showed that the strains have characteristics different from the type strains of Erwinia mallotivora, E. papayae, and E. psidii. Moreover, 16S rDNA sequence similarity searches showed that the isolated strains had less than 98.6% similarity with type strains. Additionally, phylogenetic analysis of 16S rDNA sequences suggested that the isolated strains were possibly a novel species belonging to the genus Erwinia, as the strains formed an independent cluster and had low sequence similarity with the type strains. Earlier studies indicated that papaya black rot is caused by E. cypripedii. Therefore, we propose to add the Erwinia sp. isolated in this study to the list of papaya black rot pathogens.     

香蕉枯萎病菌生理小种鉴定及其SCAR标记

 通过室内人工接种蕉类鉴别寄主,对采集于广东蕉区的18个蕉类枯萎病菌菌株进行鉴定,KP021、KP022、GZ981和JL021 4个菌株属Racel,其余14个菌株属Race4,说明广东蕉区同时存在尖孢镰刀菌古巴专化型Race1和Race4。用RAPD技术对上述18个菌株进行分析,从200条随机引物中筛选出8条引物可产生生理小种RAPD标记12个,其中标记Racel的8个,标记Race4的4个。对这些RAPD标记带分别进行回收、克隆、测序,根据这些特异片段序列分别设计相应的SCAR引物,通过对18个菌株的PCR扩增检验,有4个RAPD标记成功地转化为SCAR标记,其中Race1-SCAR标记1个、Race4-SCAR标记2个、同时能鉴定出2个小种的SCAR标记1个。应用这4个SCAR标记同时对采自田间的9个病菌分离物进行检测,能够准确地鉴定出广东蕉区的尖孢镰刀菌古巴专化型Racel和Race4,这为下一步开展香蕉枯萎病菌生理小种的分子鉴定及各生理小种田间流行动态监测奠定了基础。     

Occurrence of blood disease of banana in Sumatra,Indonesia

Since 1991, the sudden death of cultivated banana plants has been widely observed in the southern region of Sumatra Island, Indonesia. Wilting from loss of petiole and midrib turgidity, yellowing, and necrosis of leaves was followed by death of the whole plant. Reddish brown bacterial ooze exuded from the cut surface of infected pseudostems and fruits. The colony appearance of the isolated bacterium was similar to that of Ralstonia solanacearum. The bacterium was pathogenic to banana plants but not to tomato. Its bacteriological properties agreed with those of blood disease bacterium (BDB) of banana described previously. The 16S rDNA sequence of strain Banana E had conserved bases characteristic of BDB. Based on these results, the causal agent was identified as BDB, which is a close relative of Ralstonia. The isolates have resistance against antibiotics, such as chloramphenicol and tetracycline.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession number AB095535     

Characterization and genetic status of Banana bunchy top virus isolated from Okinawa, Japan

Isolates of Banana bunchy top virus (BBTV), which causes bunchy top disease in bananas, were collected in field surveys on seven islands in Okinawa Prefecture, Japan. From 44 banana samples, one isolate from each island was selected, and the DNA-1 and DNA-3 components were sequenced. Analysis of the major common region of DNA-1 showed that BBTV in Okinawa belongs to the Asian group of BBTV. DNA-1 and DNA-3 analysis revealed that Okinawan BBTV had a closer relationship with isolates from Taiwan and the Philippines than with some isolates from China and Vietnam. All the Okinawan BBTV isolates had high homology in the nucleotide sequences of DNA-1 and DNA-3 (%) because of a single, recent BBTV invasion of this area.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB108449 – AB108458     

In search of commercially acceptable Panama wilt-resistant natural variants in Ney Poovan banana (Musa AB)

Ney Poovan banana, the most widely cultivated mixed diploid banana, has been reported to be susceptible to Fusarium wilt like many other varieties in the world. The identification of natural variants possessing disease tolerance or resistance is one strategy to prevent losses. The aim of this study was to identify resistant lines in Ney Poovan banana, through extensive field surveys and screening of putative variants using a detached leaf-based challenging technique. The selected lines were screened under field condition to determine their economic feasibility for commercial-scale use. A total of 26 lines were observed to be resistant to the disease, out of which 24 exhibited a commercially acceptable productivity index and five best lines possessing desirable attributes were obtained.     

Silicon application enhances resistance to xanthomonas wilt disease in banana

Silicon (Si) has been reported to be a beneficial element and shown to enhance disease resistance in many crops, although it is not regarded to be critical for plant growth and development. In this study, the potential effect of Si supplementation on resistance to banana xanthomonas wilt (BXW) disease was evaluated using various banana cultivars. Si application at a concentration of 200 mg per plant per week was found to be optimal in enhancing resistance to BXW without any detrimental effects on plant growth. The effect of varying the duration of Si application showed continuous supply of Si before and after pathogen inoculation led to a significantly higher level of resistance to BXW in all the banana cultivars tested in comparison to non‐Si‐treated inoculated plants. Banana plants treated with Si before pathogen inoculation only, also exhibited high protection against BXW similar to plants treated continuously with Si. The total Si content in leaves increased significantly in Si‐treated plants in comparison to non‐Si‐treated control plants. The amount of Si accumulation was directly correlated to the duration of application; plants treated with Si continuously showed significantly higher amounts of Si accumulation in leaves than plants where Si application was terminated following bacterial inoculation or when Si application started immediately after pathogen inoculation. The Si‐treated plants also showed higher activity of the peroxidase enzyme in comparison to non‐Si‐treated control plants. This study confirms that application of Si enhances resistance to BXW and may provide an alternative disease management strategy.     

应用RAPD标记技术鉴定香蕉褐缘灰斑病菌

采用Mycosphaerella fijiiensis和M.musicola的RAPD标记技术鉴定海南香蕉褐缘灰斑病菌,结果表明,分离自海南儋州、乐东、文昌、东方、澄迈、临高、琼海、昌江、琼山、三亚香蕉上的10个菌株均为M.fijiensis,引起香蕉黑叶条斑病。     

Molecular diagnostics for the sigatoka disease complex of banana

ABSTRACT The Sigatoka disease complex of banana involves three related ascomycetous fungi, Mycosphaerella fijiensis, M. musicola, and M. eumusae. The exact distribution of these three species and their disease epidemiology remain unclear, because their symptoms and life cycles are rather similar. Disease diagnosis in the Mycosphaerella complex of banana is based on the presence of host symptoms and fungal fruiting structures, which hamper preventive management strategies. In the present study, we have developed rapid and robust species-specific molecular-based diagnostic tools for detection and quantification of M. fijiensis, M. musicola, and M. eumusae. Conventional species-specific polymerase chain reaction (PCR) primers were developed based on the actin gene that detected DNA at as little as 100, 1, and 10 pg/mul from M. fijiensis, M. musicola, and M. eumusae, respectively. Furthermore, TaqMan real-time quantitative PCR assays were developed based on the beta-tubulin gene and detected quantities of DNA as low as 1 pg/mul for each Mycosphaerella sp. from pure cultures and DNA at 1.6 pg/mul per milligram of dry leaf tissue for M. fijiensis that was validated using naturally infected banana leaves.     

香蕉叶鞘腐败病病原鉴定

 香蕉叶鞘腐败病为近年来发生在广东、广西、海南等地蕉园的一种新病害。经病原菌分离及柯赫氏法则检测,证明引起该病的病原菌为一种细菌,根据致病性、寄主范围测定、菌体形态、培养特性、生理生化反应及16S rDNA序列分析将该病原细菌初步鉴定为成团泛菌(Pantoea agglomerans)。成团泛菌危害香蕉,引起叶鞘腐败病在国内尚属首次报道。     

香蕉品种对假茎象甲田间抗性评价指标的研究

通过对 6个香蕉品种上的假茎象甲成、幼虫发生指数和危害程度指数调查 ,提出了香蕉品种对香蕉假茎象甲危害的田间抗性评价指标 ,用此指标评价了6个品种香蕉对假茎象甲的抗性 ,结果表明Saba大蕉、粉蕉抗虫性较强 ,龙牙蕉、香芽蕉抗虫性差     

A loop-mediated isothermal amplification (LAMP) assay based on unique markers derived from genotyping by sequencing data for rapid in planta diagnosis of Panama disease caused by Tropical Race 4 in banana

The socio-economic impact of Fusarium odoratissimum, which is colloquially called tropical race 4 (TR4), is escalating as this fungal pathogen spreads to new banana-growing areas. Hence, the development of simple, reliable and rapid detection technologies is indispensable for implementing quarantine measures. Here, a versatile loop-mediated isothermal amplification (LAMP) assay has been developed that is applicable under field and laboratory conditions. DNA markers unique to TR4 isolates were obtained by diversity arrays technology sequencing (DArTseq), a genotyping by sequencing technology that was conducted on 27 genotypes, comprising 24 previously reported vegetative compatibility groups (VCGs) and three TR4 isolates. The developed LAMP TR4 assay was successfully tested using 22 TR4 isolates and 45 non-target fungal and bacterial isolates, as well as on infected plants under greenhouse and field conditions. The detection limit was 1 pg µL⁻¹ pure TR4 DNA or 10² copies plasmid-localized TR4 unique sequence (SeqA) per reaction, which was not affected by background DNA in complex samples. The LAMP TR4 assay offers a powerful tool for the routine and unambiguous identification of banana plants infected with TR4, contributing to advanced diagnosis in field situations and monitoring of fusarium wilt.     

三氯异氰尿酸对香蕉叶鞘腐烂病的抑制效果

香蕉叶鞘腐烂病是近几年的新发病害,目前生产上尚无高效药剂,2012-2013年进行了三氯异氰尿酸对香蕉叶鞘腐烂病的抑制效果评价。两年的试验结果表明,42%三氯异氰尿酸可湿性粉剂是抑制香蕉叶鞘腐烂病的较好药剂,336倍液施药4次对香蕉叶鞘腐烂病防治效果达到63.16%、66.15%,显著优于对照药剂50%氯溴异氰尿酸可湿性粉剂348倍液施药4次后的防效,值得在香蕉产区推广应用。     

铵态氮和硝态氮对香蕉枯萎病发生的比较研究

 为寻找降低香蕉枯萎病发生的防治措施,通过室内盆栽接种试验,研究了铵态氮(NH₄⁺-N)和硝态氮(NO₃^--N)对香蕉枯萎病发生及其植株叶绿素含量、气体交换参数、病原菌在植物体内的数量分布和植物钙(Ca)、镁(Mg)、铁(Fe)、钼(Mo)、可溶性糖和木质素含量的效应。结果表明:不接种病原菌的条件下,不同氮素处理对香蕉幼苗生长影响无差异;接菌情况下,与NH₄⁺-N处理相比,NO₃^--N处理显著降低植株各器官的病原菌数量、发病率和发病严重程度。病原菌侵染后,不同氮素处理下植株光合作用均显著下降:NO₃^--N处理香蕉苗保持比NH₄⁺-N处理更高的光合速率;病原菌侵染后NH₄⁺-N处理的植株Ca、Mg、Fe和Mo含量相对于侵染前没有显著差异,但NO₃^--N处理下此4种元素含量均显著升高。病原菌侵染后的植株叶片可溶性糖含量在不同氮素处理中都没有显著变化,但在根系中,NO₃^--N处理的侵染植株可溶性糖含量显著降低。与此同时,病原菌侵染后,木质素含量在NH₄⁺-N处理植株中变化不显著,但其含量在NO₃^--N处理侵染后显著上升。综上所述,NO₃^--N处理可增加植株抗病相关矿质元素的吸收,诱导香蕉苗木质素形成,使其木质化程度增加,从而维持较高的光合作用,保持较高的抗病水平。     

Occurrence of watermark disease of willows in Japan

The watermark disease of willows was recently identified in Japan for the first time, in the northern island of Hokkaido. During spring and summer, the leaves and branches of affected trees suddenly wilted and some trees died. When affected branches or trunks were cut, a distinct, watery, reddish-brown or brownish-black stained zone (watermark) was seen in the sapwood. The causal bacterium was isolated from the watermark and identified as Erwinia salicis by its bacteriological characteristics. Inoculation tests confirmed its pathogenicity to willows.     

Identification and characterization of Colletotrichum species associated with anthracnose disease of banana

Banana (Musa spp.) is one of the five most abundantly produced fruits in the world and is widely planted in tropical and subtropical areas. Banana anthracnose is one of the main diseases during the growth and postharvest storage period of banana, seriously affecting quality and production. In this study, 24 samples of banana anthracnose were collected near the cities Nanning, Qinzhou, Baise, and Chongzuo in Guangxi Province, China. Based on colony features, conidial and appressorial morphology, and sequence analysis of several genomic regions (internal transcribed spacer [ITS] region, glyceraldehyde-3-phosphate dehydrogenase [GAPDH], actin [ACT], β-tubulin [TUB2], chitin synthase [CHS-1], calmodulin [CAL], and the intergenic region of apn2 and MAT1-2-1 [ApMAT]), the 32 Colletotrichum isolates obtained were identified as five species: C. fructicola (41%), C. cliviicola (28%), C. siamense (16%), C. karstii (9%), and C. musae (6%). A conidial suspension (10⁶ spores/ml) was used to inoculate banana seedlings for pathogenicity tests by applying 20 μl to wound sites. Lesions caused by C. musae developed most rapidly while those of C. karstii took the longest time to develop. This is the first report of C. siamense, and C. karstii associated with banana anthracnose in China, and the first report of C. fructicola and C. cliviicola associated with banana anthracnose worldwide.     

香蕉枯萎病田间发病株的高效灭菌方法筛选

为筛选有效防控香蕉枯萎病菌扩散蔓延的灭菌方法,采用含毒介质培养法测定咪鲜胺和多菌灵不同配比对枯萎病菌的毒力作用以筛选最佳混配液,比较最佳混配液与生石灰、草甘膦、咪鲜胺的平板抑菌和大田灭菌效果,并研究了大田不同施用方式对最佳混配液灭菌效果的影响。结果表明,咪鲜胺与多菌灵体积比为10∶1时得最佳混配液,可显著提高对香蕉枯萎病菌的抑制效果,增效系数为1.53,EC50值最小,为0.025 mg/L;4种药剂的EC50值由大到小为草甘膦生石灰咪鲜胺最佳混配液。1 000 mg/L最佳混配液喷施于大田病株5 d后根际土壤病菌含量下降了95.93%,10 d后球茎病菌含量减少了71.88%,综合灭菌效果显著优于其它处理。此外,应用打孔灌药法可显著提高最佳混配液对大田病株的灭菌效果,施用5 d后球茎病菌含量减少了95.95%。表明以打孔灌药法施用1 000 mg/L最佳混配液灭菌效果显著且操作便捷,易于推广应用。     

不同品种香蕉抗枯萎病效果及抗性生理研究

通过盆栽试验研究了向土壤中接种尖孢镰刀菌古巴专化型4号生理小种(FocTR4)后不同抗枯萎病香蕉品种发病率、根际可培养微生物及防御酶活性的变化。结果表明:试验处理中香蕉枯萎病发病率随着FocTR4接种浓度的增加而上升,但在相同浓度处理下,抗病品种发病率显著低于感病品种;各品种香蕉发病率与根际土壤可培养镰刀菌数量均呈显著正相关关系。抗病品种过氧化物酶(POD)、几丁质酶和β-1,3-葡聚糖酶活性高于感病品种,而与多酚氧化酶(PPO)、苯丙氨酸解氨酶(PAL)活性存在一定的相关性。说明香蕉抗病性与香蕉根际土壤微生物群落结构及香蕉本身防御酶活性有关。     

<Emphasis Type="Italic">Ageratum</Emphasis><Emphasis Type="Italic">yellow vein virus</Emphasis> isolated from tomato plants with leaf curl on Ishigaki Island,Okinawa, Japan

In 2005, severe leaf curling and yellowing were observed on tomato plants on Ishigaki Island. Because the symptoms were consistent with infection by a begomovirus, we used a polymerase chain reaction (PCR) with specific primers for begomovirus DNA-A and DNA satellite component (DNA-β) and detected products of the expected sizes from symptomatic tomato plant samples. DNA sequence analyses of the PCR products revealed that the symptomatic tomato plants were associated with Ageratum yellow vein virus (AYVV) infection. We confirmed AYVV transmission from the naturally infected weed host, Ageratum conyzoides, to healthy tomato plants by the insect vector Bemisia tabaci B biotype. This report is the first of AYVV occurrence in Japan.     

The effectiveness of consistent roguing in managing banana bunchy top disease in smallholder production in Africa

The removal of infected individuals is a common practice in the management of plant disease outbreaks. It minimizes the contact between healthy individuals and inoculum sources by reducing the infectious window of contaminated individuals. This requires early detection and consistent removal at landscape scale. Roguing of mats with symptoms of banana bunchy top disease (BBTD) in Cavendish banana production systems has been tested in Australia, using trained personnel, but has never been tested in smallholder systems. We studied the effectiveness of long-term consistent roguing in prolonging the productivity of banana orchards under smallholder farming systems in highland banana and plantain dominated production systems in Africa. We assessed the possibility of low-risk seed sourcing from the managed plots. Roguing reduced BBTD incidence to 2% in managed farmer fields and to 10% in experimental field plots, while a nonmanaged field eventually collapsed in the same period. With roguing, new infections decreased monthly compared to an exponential increase in a nonmanaged field. The emergence of new infections in both managed and nonmanaged farms followed a seasonal cycle. BBTD managed plots were a source of low-risk seed for replacing the rogued mats in the same fields, but perhaps not safe for use in nonendemic areas. We conclude that it is possible for smallholder farmers to recover and maintain banana productivity with rigorous roguing, which would entail early identification of symptoms and early removal of diseased mats. Studies are needed on the intensity of roguing under different disease and production conditions.