The use of species-specific DNA probes for the identification of Mycosphaerella fijiensis and M. musicola, the causal agents of Sigatoka disease of banana

The polymerase chain reaction (PCR)-based technique of random amplification of polymorphic DNA (RAPD) was used to differentiate DNA from species of the genus Mycosphaerella. DNA from two pathogens which cause Sigatoka leafspot diseases of banana, M. fijiensis and M. musicola , and two other Mycosphaerella species which are commonly found on banana, M. musae and M. minima , gave distinct RAPD banding patterns with all PCR primers tested. PCR, using primer RC07, amplified a 1250bp RAPD fragment from all isolates of M. fijiensis obtained from 11 geographical origins. This fragment was absent from the other species of Mycosphaerella. In Southern blots of genomic DNA, this band hybridized exclusively to DNA from M. fijiensis , and the pattern of hybridization suggested that it was binding to repeated DNA. A RAPD band amplified with primer PM06 obtained from M. musicola was also found to be species-specific. Southern analysis suggested that the fragment hybridized to a single-copy sequence in the M. musicola genome. Total genomic DNA from M. musicola was found to be a species-specific hybridization probe. Dot-blots confirmed the specificity of these probes, and could be used to identify isolates of Mycosphaerella which cause Sigatoka disease of banana in south-east Asia.     

Identification and genetic diversity of Mycosphaerella species on banana and plantain in Nigeria

Ribosomal coding DNA was sequenced and compared in 95 isolates of Mycosphaerella spp. collected in Nigeria and single nucleotide polymorphism (SNP) was used to identify the species and to determine the genetic structure of the sampled geographical populations. Using reference GenBank accessions with intercontinental distributions as controls, and shared species-specific SNPs in these control accessions, 84 (88·4%) isolates that grouped into 14 SNP haplotypes were identified as M. fijiensis , while 11 (11·6%) isolates represented by seven SNP haplotypes were characterized as M. eumusae . None of the isolates were either M. musicola or M. musae . The presence of M. fijiensis and M. eumusae in the collection was further confirmed using previously published species-specific probes designed on actin and β-tubulin gene sequences. A pairwise comparison of the population genetic distances revealed significant genetic differentiation between most populations ( P  < 0·001), with an average F _ST of 0·126, and a population structure corresponding to the four sampled geographical zones. The intraspecific dissimilarity of M. eumusae was 4·6%, compared with 2% for M. fijiensis . Compared to all the GenBank reference accessions, three sequence variations were unique to some Nigerian M. fijiensis haplotypes. Twenty-one sequence haplotypes were identified, geographically mapped and registered in GenBank. The results indicate that M. musicola has been replaced by more frequently occurring M. fijiensis and M. eumusae , against which disease management and resistance breeding efforts should be directed in Nigeria.     

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。     

Septoria Leaf Spot of Banana: A Newly Discovered Disease Caused by Mycosphaerella eumusae (Anamorph Septoria eumusae)

ABSTRACT A previously undescribed leaf spot disease of banana has been discovered in southern and Southeast Asia. The fungus identified as the causal agent of this leaf spot has a Mycosphaerella teleomorph stage and a Septoria anamorph stage. Isolation and reinoculation of the fungus to banana reproduced symptoms and confirmed its pathogenicity. Phylogenic analysis based on sequences of the internal transcribed spacer and 5.8S ribosomal DNA regions from the different leaf spot pathogens of bananas was consistent with the definition of a new species. M. eumusae (anamorph S. eumusae) is the name proposed for the causal agent and Septoria leaf spot as the name for the disease. The presence of the pathogen has been confirmed in leaf specimens from southern India, Sri Lanka, Thailand, Malaysia, Vietnam, Mauritius, and Nigeria.     

The Genetic Structure of Australian Populations of Mycosphaerella musicola Suggests Restricted Gene Flow at the Continental Scale

ABSTRACT Mycosphaerella musicola causes Sigatoka disease of banana and is endemic to Australia. The population genetic structure of M. musicola in Australia was examined by applying single-copy restriction fragment length polymorphism probes to hierarchically sampled populations collected along the Australian east coast. The 363 isolates studied were from 16 plantations at 12 sites in four different regions, and comprised 11 populations. These populations displayed moderate levels of gene diversity (H = 0.142 to 0.369) and similar levels of genotypic richness and evenness. Populations were dominated by unique genotypes, but isolates sharing the same genotype (putative clones) were detected. Genotype distribution was highly localized within each population, and the majority of putative clones were detected for isolates sampled from different sporodochia in the same lesion or different lesions on a plant. Multilocus gametic disequilibrium tests provided further evidence of a degree of clonality within the populations at the plant scale. A complex pattern of population differentiation was detected for M. musicola in Australia. Populations sampled from plantations outside the two major production areas were genetically very different to all other populations. Differentiation was much lower between populations of the two major production areas, despite their geographic separation of over 1,000 km. These results suggest low gene flow at the continental scale due to limited spore dispersal and the movement of infected plant material.     

Reactions of plantain and banana cultivars to black sigatoka leaf spot disease in three farming systems in the Nigerian guinea savanna

Journal of Plant Diseases and Protection - An assessment of the severity of black sigatoka (BS), Mycosphaerella fijiensis, leaf spot disease on plantain and banana was undertaken in three farming...     

A Quantitative Assay Using Mycelial Fragments to Assess Virulence of Mycosphaerella fijiensis

ABSTRACT We describe a method to evaluate the virulence of Mycosphaerella fijiensis, the causal agent of black leaf streak disease (BLSD) of banana and plantain. The method is based on the delivery of weighed slurries of fragmented mycelia by camel's hair brush to 5-by-5-cm areas on the abaxial surface of banana leaf blades. Reliable BLSD development was attained in an environmental growth chamber with stringent lighting and humidity controls. By localizing inoculum onto small areas of large leaves, we achieved a dramatic increase in the number of strains that can be tested on each leaf and plant, which is critical for comparing the virulence of numerous strains concurrently. Image analysis software was used to measure the percentage of each inoculated leaf section showing BLSD symptoms over time. We demonstrated that the level of disease of four isolates was correlated with the weight of the mycelium applied and relatively insensitive to the degree of fragmentation of hyphae. This is the first report demonstrating that weighed mycelial inoculum, combined with image analysis software to measure disease severity, can be used to quantitatively assess the virulence of M. fijiensis under rigorously controlled environmental conditions.     

Population differentiation in the banana leaf spot pathogen Mycosphaerella musicola, examined at a global scale

Single-copy restriction fragment length polymorphism (RFLP) markers were used to determine the genetic structure of the global population of Mycosphaerella musicola , the cause of Sigatoka (yellow Sigatoka) disease of banana. The isolates of M. musicola examined were grouped into four geographic populations representing Africa, Latin America and the Caribbean, Australia and Indonesia. Moderate levels of genetic diversity were observed for most of the populations ( H  = 0·22–0·44). The greatest genetic diversity was found in the Indonesian population ( H =  0·44). Genotypic diversity was close to 50% in all populations. Population differentiation tests showed that the geographic populations of Africa, Latin America and the Caribbean, Australia and Indonesia were genetically different populations. Using F _ST tests, very high levels of genetic differentiation were detected between all the population pairs ( F _ST > 0·40), with the exception of the Africa and Latin America-Caribbean population pair. These two populations differed by only 3% ( F _ST = 0·03), and were significantly different ( P  < 0·05) from all other population pairs. The high level of genetic diversity detected in Indonesia in comparison to the other populations provides some support for the theory that M. musicola originated in South-east Asia and that M. musicola populations in other regions were founded by isolates from the South-east Asian region. The results also suggest the migration of M. musicola between Africa and the Latin America-Caribbean region.     

Black leaf streak (Mycosphaerella fijiensis) in Uganda

The occurrence of black leaf streak caused by Mycosphaerella fijiensis is reported on a number of banana cultivars in Uganda, demonstrating the spread of the disease from West and Central to East Africa since the early 1980s.     

Dynamics of QoI Sensitivity in Mycosphaerella fijiensis in Costa Rica During 2000 to 2003

ABSTRACT From 1997 onward, the strobilurin fungicide azoxystrobin was widely used in the main banana-production zone in Costa Rica against Mycosphaerella fijiensis var. difformis causing black Sigatoka of banana. By 2000, isolates of M. fijiensis with resistance to the quinolene oxidase inhibitor fungicides were common on some farms in the area. The cause was a single point mutation from glycine to alanine in the fungal target protein, cytochrome b gene. An amplification refractory mutation system Scorpion quantitative polymerase chain reaction assay was developed and used to determine the frequency of G143A allele in samples of M. fijiensis. Two hierarchical surveys of spatial variability, in 2001 and 2002, found no significant variation in frequency on spatial scales <10 m. This allowed the frequency of G143A alleles on a farm to be estimated efficiently by averaging single samples taken at two fixed locations. The frequency of G143A allele in bulk samples from 11 farms throughout Costa Rica was determined at 2-month intervals. There was no direct relationship between the number of spray applications and the frequency of G143A on individual farms. Instead, the frequency converged toward regional averages, presumably due to the large-scale mixing of ascospores dispersed by wind. Using trap plants in an area remote from the main producing area, immigration of resistant ascospores was detected as far as 6 km away both with and against the prevailing wind.     

香蕉炭疽菌rDNA ITS区的分子鉴定与检测

 香蕉炭疽病菌(Colletordchum muscat)是一种引起香蕉采后病害的最重要病原,本研究用真菌18S~28S间的内转录间隔区(internal transcribed spacer,ITS)通用引物18SF和28SR扩增香蕉炭疽菌和其它外群真菌的基因组DNA,扩增出约510bp的片段;通过克隆测序香蕉炭疽菌的ITS全序列并与GenBank中炭疽菌属其它种的ITS序列比对,设计出香蕉炭疽菌的特异性引物ColM1和ColM2。用此特异引物可以从香蕉炭疽菌株中扩增出382bp的特异性片段,而其余20个参试菌株和香蕉组织的PCR反应结果为阴性,灵敏度实验证明可以检测到目标DNA的浓度为0.1Pg。该方法可用于快速、准确和灵敏地检测香蕉炭疽菌,为快速监测组织中有无香蕉炭疽病菌潜伏侵染与及早采取防治措施提供积极的指导意义。     

Development of Nested Polymerase Chain Reaction Detection of Mycosphaerella spp. and Its Application to the Study of Leaf Disease in Eucalyptus Plantations

ABSTRACT Mycosphaerella leaf disease (MLD) is a serious disease of two of the major eucalypt species grown in temperate regions worldwide, Eucalyptus globulus and E. nitens. More than 30 species of Mycosphaerella have been reported on eucalypts worldwide. Accurate, rapid, and early discrimination of Mycosphaerella spp. causing crown damage to E. globulus and E. nitens will assist the development of sustainable management strategies. This study describes the development, and incorporation in a nested polymerase chain reaction (PCR) approach, of specific primers for the detection and identification of Mycosphaerella spp. commonly reported from leaf lesions of E. globulus and E. nitens in Australia. Primer design was assisted by sequence alignment and phylogenetic analysis of 165 nonredundant sequences from the nuclear ribosomal DNA internal transcribed spacer regions of Mycosphaerella and related species. Phylo-genetic analysis revealed very high sequence similarity for two taxon groups, Mycosphaerella grandis and M. parva, and M. vespa, M. ambi phylla, and M. molleriana, and primers were designed to differentiate each of the two groups. Three other species, M. cryptica, M. nubilosa, and M. tasmaniensis, were distinct and distinguished by species-specific primers. In double-blind trials, the detection test accurately and rapidly identified Mycosphaerella spp. in cultures and discriminated against other pathogens that co-occur in or on Eucalyptus leaves, thereby verifying its reliability. The detection test has an internal amplification control in the first-round PCR with fungal-specific primers to raise confidence in test results, particularly to highlight negative results due to PCR inhibition. When applied to DNA extracted from leaf or stem samples either as multiple or single lesions, it detected and identified up to five Mycosphaerella spp. or taxon groups in both positively identified and in young (putative) MLD lesions. The samples were 20 mm(2) or larger in surface area and were collected while undertaking disease rating assessments in an experimental investigation of Eucalyptus plantations and regrowth forest. Using nested PCR detection, Mycosphaerella spp. were positively identified in 2 days, 1 to 5 months earlier than by classical methods, demonstrating the potential application of this detection test to the early discrimination of MLD components in ecological, epidemiological, and genetic investigations.     

Genetic structure of Mycosphaerella fijiensis populations from Australia, Papua New Guinea and the Pacific Islands

Single-copy restriction fragment length polymorphism (RFLP) markers were used to determine the genetic structure of Mycosphaerella fijiensis , the cause of black leaf streak (black Sigatoka) disease of banana and plantain, in the Torres Strait, Papua New Guinea (PNG), and the Pacific Islands. A moderate level of genetic variation was observed in all populations with genotypic diversity values of 60–78% of the theoretical maximum, and gene diversity ( H ) values between 0·269 and 0·336. All populations were at gametic equilibrium, and with the high level of genotypic diversity observed this indicated that sexual reproduction has a major role in the genetic structure of the M. fijiensis populations examined. Population differentiation was tested on several hierarchical scales. No evidence of population differentiation was observed between sites on Mer Island. A moderate level of population differentiation was observed within the Torres Strait, between Badu and Mer Islands ( F _ST = 0·097). On a regional scale, the greatest differentiation was found between the populations of the Torres Strait and the Pacific. Populations from these regions were more closely related to the PNG population than to each other, suggesting they were founded in separate events from the same population.     

香蕉黑腐病菌(Botryodiplodia theobromae)的PCR检测

 根据香蕉黑腐病菌可可球二孢菌(Botryodiplodia theobromae)与其它香蕉病原真菌核糖体基因转录间隔区(rDNA-ITS)ITS1和ITS2间序列差异,设计了特异引物Bth-S(5'-TCTCCCACCCTTTGTGAAC-3')和Bth-A(5'-AAAAGT-TCAGAAGGTTCGTC-3'),利用此引物对包括可可球二孢菌在内的21个菌株基因组DNA进行PCR扩增,结果只有4个可可球二孢菌菌株扩增到422bp特异带,其它17个菌株无扩增产物。灵敏度测试结果表明此特异引物能对1pg的可可球二孢菌基因组DNA进行扩增。对自然感染黑腐病的香蕉果实组织和接种可可球二孢菌或多种香蕉病原真菌混合接种的果实组织进行检测,Bth-S和Bth-A引物对不仅能够在自然感染黑腐病果实组织中特异检测到可可球二孢菌,而且能在未显症和发病的接菌香蕉果实组织中特异检测得到可可球二孢菌。这为香蕉可可球二孢菌潜伏侵染检测提供了技术支持。     

基于Ypt1基因为靶标的木香疫病病组织及病田土壤分子检测技术

A species-specific PCR assay was established for rapid and accurate detection of the oomycete pathogen Phytophthora tentaculata in diseased plant tissues and infected soil.A pair of species-specific primers Pt1/Pt2 were designed on the basis of Ras-related protein(Ypt1) gene sequences of the Phytophthora species.PCR amplification with the Pt primers resulted in a 386 bp product only from isolates of P.tentaculata.The detection threshold with Pt primers was 100 pg of genomic DNA.A nested PCR procedure was developed using Ypt1F/Ypt1R as the first-round amplification primers and Pt1/Pt2 as the second-round primers,which increased the detection sensitivity 100-fold to 1 pg.PCR using these Pt primers can also be used to detect P.tentaculata in naturally infected plant tissues and soil.The PCR-based method developed in this study provides a rapid and sensitive tool for detection of P.tentaculata.     

Molecular detection of Phytophthora capsici in infected plant tissues, soil and water

A species-specific PCR assay was developed for rapid and accurate detection of the pathogenic oomycete Phytophthora capsici in diseased plant tissues, soil and artificially infested irrigation water. Based on differences in internal transcribed spacer (ITS) sequences of Phytophthora spp. and other oomycetes, one pair of species-specific primers, PC-1/PC-2, was synthesized. After screening 15 isolates of P. capsici and 77 isolates from the Ascomycota, Basidiomycota, Deuteromycota and Oomycota, the PC-1/PC-2 primers amplified only a single PCR band of c . 560 bp from P. capsici . The detection sensitivity with primers PC-1/PC-2 was 1 pg genomic DNA (equivalent to half the genomic DNA of a single zoospore) per 25- µ L PCR reaction volume; traditional PCR could detect P. capsici in naturally infected plant tissues, diseased field soil and artificially inoculated irrigation water. Using ITS1/ITS4 as the first-round primers and PC-1/PC-2 in the second round, nested PCR procedures were developed, increasing detection sensitivity to 1 fg per 25- µ L reaction volume. The results suggested that the assay detected the pathogen more rapidly and accurately than standard isolation methods. The PCR-based methods developed here could simplify both plant disease diagnosis and pathogen monitoring, as well as guiding plant disease management.     

A Species-Specific Polymerase Chain Reaction Assay for Rapid Detection of Phytophthora nicotianae in Irrigation Water

ABSTRACT Phytophthora nicotianae is a common and destructive pathogen of numerous ornamental, agronomic, and horticultural crops such as tobacco, tomato, and citrus. We have developed a species-specific polymerase chain reaction (PCR) assay for rapid and accurate detection of this pathogen in irrigation water, a primary source of inoculum and an efficient means of propagule dissemination. This PCR assay consists of a pair of species-specific primers (PN), customization of a commercial soil DNA extraction kit for purification of DNA from propagules in irrigation water, and efficient PCR protocols for primer tests and sample detection. The PN primers proved adequately specific for P. nicotianae in evaluations with 131 isolates of P. nicotianae, 102 isolates from 15 other species of Phytophthora, and 64 isolates from a variety of other oomycetes, true fungi, and bacteria. These isolates originated from a wide range of host plants, three substrates (plant tissue, soil, and irrigation water), and numerous geographic locations. The detection sensitivity is between 80 and 800 fg DNA/mul. The assay detected the pathogen in naturally infested water samples from Virginia and South Carolina nurseries more rapidly and accurately than standard isolation methods. Use of this PCR assay can assist growers in making timely disease management decisions with confidence.     

玉米圆斑病病原的快速检测

玉米生平脐蠕孢菌(Bipolaris zeicola)是引起玉米圆斑病的病原菌。本研究通过对玉米生平脐蠕孢菌及其近似种的EF-1α基因(elongation factor 1α)部分序列进行比对,设计出玉米生平脐蠕孢菌的特异性引物Y-EF-F和Y-EF-R,利用该引物可以从B.zeicola中扩增出137 bp的特异片段,而其余的17个参试菌株扩增结果为阴性。灵敏度实验表明该对引物可以检测到目标DNA的浓度为1 pg·μL~(-1)。用B.zeicola接种玉米叶片、苞叶以及玉米粒,然后以接种发病的病组织DNA为模板,利用引物Y-EF-F和Y-EF-R进行PCR扩增,可以扩增出137 bp的特异性条带,而健康玉米组织DNA中未能扩增出任何条带。用B.zeicola孢子悬浮液接种大田玉米叶片,接种第3 d可以检测到未发病组织中有B.zeicola病原菌,第5 d可以看到明显的病斑。研究结果表明该方法可用于快速、准确和灵敏地检测玉米组织中的潜伏期玉米生平脐蠕孢菌,为玉米圆斑病的快速检测,进而及早采取防治措施提供积极的指导。     

A nested-polymerase chain reaction protocol for the detection of Mycosphaerella nawae in persimmon

Mycosphaerella nawae is the causal agent of circular leaf spot of persimmon. A polymerase chain reaction (PCR) based protocol was developed for M. nawae-specific identification from pure culture, or infected symptomatic and asymptomatic persimmon tissues. Variation among the internal transcribed spacer regions (ITS) of the ribosomal DNA (rDNA) sequences of potentially related fungal species in persimmon orchards was analyzed for a primer pair design. Specificity was confirmed using multiple isolates of these species, other fungal pathogens that cause foliar diseases in persimmon and contaminants commonly obtained in the isolation process. The detection threshold for M. nawae DNA was lowered from 50 pg to 500 fg when nested-PCR was evaluated instead of single PCR. The nested-PCR protocol developed in this study showed its suitability to be applied for the specific detection of M. nawae from three types of naturally infected persimmon tissues: from lesions in fresh leaves, from pseudothecia present in lesions in leaf litter, and from infected asymptomatic leaves. The protocol can be useful for routine diagnosis, disease monitoring programs and for epidemiological research.     

Development and application of a PCR-based 'molecular tool box' for the identification of Phytophthora species damaging forests and natural ecosystems

A PCR-based 'molecular tool box', based on a region of the ras-related protein gene Ypt 1, was developed for the identification of 15 Phytophthora species that damage forests and trees: P. cactorum , P. cambivora , P. cinnamomi , P. citricola , P. europaea , P. inundata , P. lateralis , P. megasperma , P. nemorosa , P. kernoviae , P. pseudosyringae , P. psychrophila , P. quercina , P. ramorum and P. ilicis . Most primers proved highly specific in blast analyses and in tests with DNA from 72 isolates of 35 species of Phytophthora and nine species representative of Pythium . Exceptions were primers designed for P. cactorum and P. ilicis , which cross-reacted with P. idaei and P. nemorosa , respectively. Amplification with Phytophthora -genus-specific primers before amplification with the various species-specific primers (nested PCR) increased the sensitivity of detection over amplification with species-specific primers only: detection limits ranged between 100 and 10 pg target DNA µ L⁻¹ in the latter, compared with 100 fg µ L⁻¹ in nested PCR. Using existing methods for rapid extraction and purification of DNA, single-round amplification was appropriate for detection of target Phytophthora species in leaves, but nested PCR was required for soil and water samples. The quarantine pathogens P. ramorum and P. kernoviae were detected in a number of naturally infected leaves collected in England and Wales, whereas P. citricola was commonest in water and soil samples from natural Scottish ecosystems.