RAPD cluster analysis and chlorate sensitivity of some Indian isolates of Macrophomina phaseolina from sorghum and their relationships with pathogenicity

Charcoal rot caused by Macrophomina phaseolina is an economically important disease in sorghum grown during the post rainy season in India. Variations in random amplified polymorphic DNA (RAPD) polymorphisms, chlorate sensitivity and pathogenicity were studied among sorghum isolates of M. phaseolina collected from different parts of India. RAPD data based on 14 random primers of Kit A and C (OPA and OPC) on 20 isolates showed a high degree of polymorphism (98.1%) in different isolates. UPGMA dendrogram on RAPD data produced 7 clusters at the level of 37% similarity. Isolates from the same locations showed a tendency to group closer, substantiating closer genetic relatedness. Sorghum infecting Macrophomina isolates showed a mixed response for sensitivity to potassium chlorate (120 mM). Chlorate-resistant isolates were predominant (>65% of the isolates) over sensitive isolates. Chlorate-sensitive isolates were found to be genetically closer among them than the resistant ones. For the first time it was shown that chlorate sensitivity in Macrophomina had some relations with charcoal rot severity in sorghum.     

Glutamine Synthetase Activity, Relative Water Content and Water Potential in Maize Submitted to Drought

Primer screening and optimization for random amplified polymorphic DNA (RAPD) analysis of cashew (Anacardium occidentale L.) was investigated. Among four series (A, B, D and N) of 10-mer primers, A-series performed better amplification of fragments than other series. The maximum amplification fragments was obtained using OPA-02, OPA-03, OPA-09, OPB-06, OPB-10, OPD-03, OPD-05 and OPN-03 primers. The primers OPA-02 and OPN-03 produced maximum number of DNA fragments in Anacardium occidentale cv. H-320. Primers (OPB-08 and OPN-05 performed a least number of amplification fragments. RAPD profile also indicate that some primer did not produce good amplification. The primer OPA-02 amplified 12 number of polymorphic bands in 20 cultivars of cashew. Only one DNA fragment was produced in A. occidentale cv. Vridhachalam - 2 (M-44/3) by using the primer OPA-02. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic differentiation of charcoal rot pathogen, Macrophomina phaseolina, into specific groups using URP-PCR

Forty isolates of Macrophomina phaseolina, a pathogen causing charcoal dry root rot of soybean, cotton, and chickpea, were genetically characterized with universal rice primers (URP; primers derived from DNA repeat sequences in the rice genome) using polymerase chain reaction (URP-PCR). Out of 12 URPs used in this study, 5 primers were effective in producing polymorphic fingerprint patterns from the DNA of M. phaseolina isolates. Three primers (URP-2F, URP-6R, and URP-30F) were quite informative and produced high levels of polymorphism among the isolates of M. phaseolina. Analysis of the entire fingerprint profiles using unweighted pair-group method with arithmetic averages (UPGMA) clearly differentiated M. phaseolina isolates obtained from soybean, cotton, and chickpea hosts into specific groups. In this study, we found for the first time transferability and use of PCR primers derived from plant genomes to generate host-specific fingerprint profiles of M. phaseolina, a broad host range plant pathogenic fungus. These results demonstrate that URPs are sensitive and technically simple to use for assaying genetic variability in M. phaseolina populations.     

Differentiation ofFusarium sambucinum Fuckel sensu lato and related species by RAPD PCR

DNA polymorphisms generated by the random amplified polymorphic DNA polymerase chain reaction (RAPD PCR) were used to analyse 41 isolates investigated in the EuropeanFusarium sambucinum Project (EFSP). Employing ten arbitrary (10-mer) oligonucleotides and simple repeat sequences (M13, (GACA)₄) as single primers, informative banding patterns typical for identifying European populations ofFusarium sambucinum Fuckel s. str.,F. torulosum (Berk. & Curt.) Nirenberg andF. venenatum Nirenberg were obtained.     

Strain-specific detection of introduced Beauveria bassiana in agricultural fields by use of sequence-characterized amplified region markers

Field studies on the efficacy and persistence of an introduced strain of Beauveria bassiana for insect control require detection assays to differentiate the non-native strain from indigenous populations. In this study we developed strain-specific molecular markers based on polymerase chain reaction amplification of sequence-characterized amplified regions (SCAR) in combination with dilution plating on semi-selective medium to detect and estimate density of propagules of a commercial strain of B. bassiana (strain GHA) in field samples. Using random amplified polymorphic DNA (RAPD) analysis, unique fragments that distinguished GHA from other strains of B. bassiana were obtained. Three amplicons, OPA-14(0.44), OPA-15(0.44), and OPB-9(0.67), generated with RAPD primers were cloned and sequenced and used as bases for designing SCAR primers OPA14 F/R(445), OPA15 F/R(441), and OPB9 F/R(677), respectively. All three SCAR primers were highly sensitive, capable of detecting 100pg B. bassiana GHA genomic DNA, and thus could be used to detect varying levels of the fungus in the field.     

Molecular characterisation of Fusarium oxysporum causing rot diseases in small cardamom (Elettaria cardamomum Maton)

Incidence of root rot and foliar yellowing, rhizome rot, panicle wilt and stem rot diseases of small cardamom (Elettaria cardamomum Maton) are caused by Fusarium oxysporum Schlecht., and were surveyed in the high ranges of Idukki district, Kerala during 2010–2011. The diseases were noticed in different areas to varying degrees. Root rot was found to be most severe, followed by pseudostem rot, rhizome rot and panicle wilt. The Fusarium infections were prevalent throughout the year (January–December) and varied from 1.5 to 10.6%. Even though the pathogen was isolated from different plant parts, during pathogenicity studies, all the isolates could cross-infect other plant parts too. Twenty different isolates of F. oxysporum were obtained from diseased samples, and five morphologically distinct isolates were analysed with Randomly Amplified Polymorphic DNA (RAPD) markers to study the genetic variability, if any, among them. PCR amplification of total genomic DNA with random oligonucleotide primers generated unique banding patterns, depending upon primers and isolates. Nine oligunucleotide primers were selected for the RAPD assays, which resulted in 221 bands for the five isolates of F. oxysporum. The number of bands obtained was entered into an NTSYS, and the results showed moderate genetic variability among F. oxysporum isolates causing root rot, rhizome rot, panicle wilt and pseudostem rot, collected from different locations. The dendrogram of different isolates into groups resulted in one major cluster at 0.61 similarity index comprising of four isolates (CRT 3, CRR 3, CPW 2 and CSR 1) and one isolate (CRT 5) formed in a separate cluster. Among the five isolates of F. oxysporum, CRT 5 was entirely different from the other four isolates. The isolates also differ according to the geographical area, as revealed from the genetic variability observed in different root rot isolates (CRT 3 and CRT 5). It is inferred that despite moderate variability, F. oxysporum, infecting small cardamom in Idukki district of Kerala, consists of a single clonal lineage.     

DNA fingerprinting of Peronospora parasitica,a biotrophic fungal pathogen of crucifers

The fungus Peronospora parasitica (Pers. ex Fr.) Fr. is an obligate biotroph infecting a wide range of host species in the family Cruciferae. Isolates from different hosts are morphologically similar, and pathotypes are usually distinguished on the basis of host range. Random Amplified Polymorphic DNA (RAPD) fingerprints were generated from a range of P. parasitica isolates from different Brassica species. Reaction conditions, in particular DNA template, primer and Mg²⁺ concentrations, were optimized to ensure that amplifications were reproducible. Possible artefacts arising through host plant DNA were assessed by including such DNA in control reactions. Confirmation that diagnostic RAPD bands were generated from fungal DNA was also obtained by Southern hybridization of a RAPD band to genomic fungal DNA. By screening 20 decamer primers, 2 were found to detect sufficient genetic variation to allow complete differentiation between pathotypes. These results illustrate the potential value of RAPDs for detecting polymorphisms between isolates of a non-culturable plant pathogenic fungus.     

Diversity among clinical isolates of Histoplasma capsulatum detected by polymerase chain reaction with arbitrary primers.

Clinical isolates of the fungal respiratory and systemic pathogen Histoplasma capsulatum have been placed in several different classes by using genomic restriction fragment length polymorphisms (RFLPs), but in general have not been distinguished further. We report here that a polymerase chain reaction (PCR)-based DNA fingerprinting method that has been termed arbitrary primer or random amplified polymorphic DNA (RAPD) PCR can distinguish among isolates in a single RFLP class. In this method, arbitrarily chosen oligonucleotides are used to prime DNA synthesis from genomic sites that they fortuitously match, or almost match, to generate strain-specific arrays of DNA fragments. Each of 29 isolates of RFLP class 2, the group endemic in the American Midwest, was distinguished by using just three arbitrary primers. In contrast, laboratory-derived S and E colony morphology variants of two strains were not distinguished from their R parents by using 18 such primers. Thus, the clinical isolates of H. capsulatum are quite diverse, but their genomes remain stable during laboratory culture. These outcomes suggest new possibilities for epidemiological analysis and studies of fungal populations in infected hosts.     

Quick and accurate detection of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">carthami</Emphasis> in host tissue and soil using conventional and real-time PCR assay

Safflower wilt, caused by Fusarium oxysporum f. sp. carthami (Foc) is a major limiting factor for safflower (Carthamus tinctorius) production worldwide. In India alone, about 40–80% disease incidence has been reported. A rapid, efficient, specific, and sensitive diagnostic technique for Foc is therefore crucial to manage Fusarium wilt of safflower. Twenty-five isolates of F. oxysporum formae speciales infecting other crops, 17 isolates of Fusarium spp. and seven isolates of other fungal pathogens of safflower along with 75 Foc isolates were used for identification of band specific to Foc using inter-simple sequence repeat (ISSR) analysis. Out of 70 ISSR primers, the one that specifically amplified a 490 bp fragment from all the Foc isolates was selected. Sequence of the amplified fragment was utilized to design sequence characterized amplified region (SCAR) primers (FocScF/FocScR). The primer pair unambiguously and exclusively amplified a DNA fragment of approximately 213 bp in all the 75 Foc isolates. The primer set was able to detect as low as 10 pg of Foc genomic DNA using conventional PCR, while the SCAR primers when coupled with real-time qPCR demonstrated detection limits of 1 pg for Foc genomic DNA and 1000 conidia/g for soil. The assay enabled reliable diagnosis of Foc DNA in contaminated safflower fields and expedited Foc detection at 72 h post inoculation in asymptomatic seedlings. This method facilitates quick and precise detection of Foc in plant and soil samples and can be exploited for timely surveillance and sustainable management of the disease.     

Genetic Variability of Fusarium oxysporum f.sp. ciceris Population Affecting Chickpea in the Sudan

Fusarium wilt caused by Fusarium oxysporum f.sp. ciceris (Foc) is the most important soilborne disease of chickpea in the Sudan and many other countries. A total of 76 Foc isolates from six different chickpea‐growing states in the Sudan have been collected in this study to investigate the genetic diversity of Sudanese Foc isolates. Additional 14 Foc isolates from Syria and Lebanon were included in this study. All isolates were characterized using four random amplified polymorphic DNA (RAPD), three simple sequence repeats (SSR), five sequence‐characterized amplified region (SCAR) primers and three specific Foc genome primers. Based on the similarity coefficient, the results indicated two major clusters included seven subclusters. The isolates from the Sudan were grouped as identified as races 0, 2 and unknown races. The isolates from Syria and Lebanon were grouped together as they identified as races 1B/C and 6, respectively. This study identified a new race Foc (race 0) in the Sudan. The results of this study will be useful for breeders to design effective resistance breeding program in chickpea in the Sudan.     

Application of RAPD analysis for identification of Lactococcus lactis subsp. cremoris strains isolated from artisanal cultures

Randomly amplified polymorphic DNA (RAPD) was used for identification of Lactococcus lactis subsp. cremoris strains isolated 40 years ago from various dairy homemade products. Total genomic DNAs from six randomly chosen isolates and the reference strain Lactococcus lactis subsp. cremoris NIZO B64 were amplified using four different 10-mer primers. Although most RAPD fragments were common to all six isolates, a sufficient number of polymorphic fragments were also detected that allowed clear distinction of the isolates and the reference strain. The results indicate that RAPD analysis could be a useful and efficient method to distinguish Lactococcus lactis subsp. cremoris at the strain level and to detect genetic diversity.     

黑龙江省葫芦科白粉病菌RAPD分析

2010年采集黑龙江省不同生态区不同设施内的甜瓜、黄瓜、南瓜、西瓜等瓜类白粉病菌菌株17份,采用国际通用的瓜类白粉病菌生理小种鉴别寄主对17份白粉病菌进行了生理小种鉴定。根据13个鉴别寄主的抗感反应,初步确定黑龙江省葫芦科作物白粉病菌存在3个生理小种,即单囊壳白粉菌Podosphaera xanthii的生理小种1和生理小种N1号及一个新生理小种,其中生理小种1为优势小种。通过对13份白粉病菌的RAPD分析,从119个随机引物中筛选出10个条带清晰而且重复性好的引物,扩增得到157个位点,其中多态性位点为138个,多态性位点频率为97.89%,表明黑龙江省葫芦科作物白粉菌具有丰富的遗传多样性。利用NTSYS-PC软件进行数据分析,结果表明13个菌株之间遗传相似系数的变化幅度为0.52－0.75。根据遗传相似系数用类平均法（UPGMA）对其聚类,以遗传相似系数0.60为阈值,供试菌株可区分为4个类群。同是生理小种1的菌株部分聚到了同一类,新生理小种与部分生理小种1菌株聚到同一类,同是生理小种N1的两个菌株未聚到同一类;相同地理来源或相同寄主来源的白粉菌也未聚到一类。初步确定葫芦科白粉病菌致病性与DNA多态性不形成对应关系,菌株的遗传多样性与菌株地理来源、寄主来源及设施类型亦无明显的直接关系。     

Differentiation of <Emphasis Type="Italic">Aspergillus niger</Emphasis> by random amplification of polymorphic DNA

The randomly amplified polymorphic DNA (RAPD) patterns of whole-cell lysates from five Aspergillus niger isolates, including one reference strain, two isolated from deep freeze, and two environmental strains from soil and plant infections, were investigated. PCR-RAPD analysis of genomic DNA was performed using eight primers (Tube-A1, Tube-A6, Tube-A17, Tube-B8, Tube-B11, Tube-B15, Tube-C5, Tube-C6). The RAPD assay discriminated between all strains. Comparison of deep freeze isolates showed identical RAPD patterns in some of the reference and environmental isolates. The data indicates that the RAPD technique is useful for fingerprinting A. niger.     

基于RAPD、ISSR和AFLP对西瓜枯萎病菌遗传多样性的评价

利用RAPD、ISSR和AFLP分子标记技术对50个西瓜枯萎病菌株进行了分析。结果表明,21个RAPD引物、21个ISSR引物和21对AFLP引物分别对供试菌株扩增出113、134和389条带,三种分子标记的遗传相似系数比较一致,均可揭示西瓜枯萎病菌的遗传变异特点。三种分子标记产生的聚类分析结果存在一定差异,其中RAPD类群与生理小种和地理来源之间均不存在明显关系;而AFLP和ISSR类群与生理小种之间存在一定相关性,与菌株的地理来源关系不明显。     

基于RAPD、ISSR和AFLP对西瓜枯萎病菌遗传多样性的评价

利用RAPD、ISSR和AFLP分子标记技术对50个西瓜枯萎病菌株进行了分析。结果表明,21个RAPD引物、21个ISSR引物和21对AFLP引物分别对供试菌株扩增出113、134和389条带,三种分子标记的遗传相似系数比较一致,均可揭示西瓜枯萎病菌的遗传变异特点。三种分子标记产生的聚类分析结果存在一定差异,其中RAPD类群与生理小种和地理来源之间均不存在明显关系;而AFLP和ISSR类群与生理小种之间存在一定相关性,与菌株的地理来源关系不明显。     

Identification and typing of food-borne Staphylococcus aureus by PCR-based techniques

The possibility of using PCR for rapid identification of food-borne Staphylococcus aureus isolates was evaluated as an alternative to the API-Staph system. A total of 158 strains, 15 S. aureus, 12 other staphylococcal species, and 131 isolates recovered from 164 food samples were studied. They were phenotypically characterized by API-Staph profiles and tested for PCR amplification with specific primers directed to thermonuclease (nuc) and enterotoxin (sea to see) genes. Disagreement between the PCR results and API-Staph identification was further assessed by the analysis of randomly amplified polymorphic DNA (RAPD) profiles obtained with three universal primers (M13, T3, and T7) and 16S rDNA sequencing. Forty out of 131 isolates (31%) tested positive for PCR enterotoxin. Of these, 14 (11%) were positive for sea, 22 (17%) for sec, one (0.8%) for sed, and three (2.2%) for sea and sec. No amplification corresponding to seb nor see was obtained. Cluster analysis based on RAPD profiles revealed that most of the sec positive food isolates grouped together in three clusters. Cluster analysis combining the three RAPD fingerprints (M 13, T3, and T7), PCR-enterotoxin genotype and API-Staph profiles, grouped the nuc PCR positive isolates together with S. aureus reference strains and the nuc PCR negative isolates with reference strains of other staphylococcal species. The only nuc PCR positive food isolate that remained unclustered was a sed positive strain identified by 16S rDNA sequence as S. simulans. The high concordance between S. aureus and nuc PCR positive strains (99%) corroborates the specificity of the primers used and the suitability of nuc PCR for rapid identification of S. aureus in routine food analysis.     

海南省香蕉枯萎菌生理小种的RAPD分析

利用随机扩增多态性DNA(RAPD)分子标记方法对海南省香蕉枯萎病菌2个生理小种(小种1和小种4)进行遗传多样性分析,以筛选出的15个随机引物对采自海南省各市县发病蕉区的分别属于1号生理小种和4号生理小种的16个代表菌株及广东省2个1号和4号生理小种对照菌株进行RAPD-PCR扩增,结果产生97个RAPD分子标记,其中多态性的条带有76条,通过聚类分析探讨了供试小种间的亲缘关系,并寻找到了1、4号生理小种的特异性条带,为在分子水平上进行香蕉枯萎病菌生理小种鉴定提供更为便利的手段。     

Random amplified polymorphic DNA (RAPD) identification of genetic variation in three species ofPorphyra (Bangiales,Rhodophyta)

The random amplified polymorphic DNA (RAPD) technique was used to characterize three species ofPorphyra from the western North Atlantic and adjacent Gulf of Mexico. Twenty 10-mer primers were screened for DNA amplification usingPorphyra template DNA. Nine of these oligonucleotide primers, all (G+C)-rich, were positive or band-producing, but yielded poor or variable band resolution. Subsequent use of the universal 20-mer M 13 primer resulted in both clear band resolution with a minimum of secondary bands and a high degree of reproducibility. Amplification products for DNA from six regional isolates ofPorphyra carolinensis Coll et Cox,P. leucosticta Thuret in Le Jolis andP. rosengurttii Coll et Cox were compared to each other and toBangia atropurpurea (Roth) C. Agardh. Results provide evidence of both genetically hetero- and homogeneous populations. Use of the RAPD method with the M 13 primer yields amplification products which can be used to fingerprint specific genotypes. This procedure could be used to discriminate between hetero- and homokaryotic fusion products from previously characterized donor strains.     

Molecular detection and genotyping of Fusarium oxysporum f. sp. psidii isolates from different agro-ecological regions of India

Twenty one isolates of Fusarium oxysporum f. sp. psidii (Fop), causing a vascular wilt in guava (Psidium guajava L.), were collected from different agro-ecological regions of India. The pathogenicity test was performed in guava seedlings, where the Fop isolates were found to be highly pathogenic. All 21 isolates were confirmed as F. oxysporum f. sp. psidii by a newly developed, species-specific primer against the conserved regions of 28S rDNA and the intergenic spacer region. RAPD and PCR-RFLP were used for genotyping the isolates to determine their genetic relationships. Fifteen RAPD primers were tested, of which five primers produced prominent, polymorphic, and reproducible bands. RAPD yielded an average of 6.5 polymorphic bands per primer, with the amplified DNA fragments ranging from 200–2,000 bp in size. A dendrogram constructed from these data indicated a 22–74% level of homology. In RFLP analysis, two major bands (350 and 220 bp) were commonly present in all isolates of F. oxysporum. These findings provide new insight for rapid, specific, and sensitive disease diagnosis. However, genotyping could be useful in strain-level discrimination of isolates from different agro-ecological regions of India.     

Genomic fingerprinting of Bradyrhizobium japonicum isolates by RAPD and rep-PCR

Genetic diversity of indigenous Bradyrhizobium japonicum population in Croatia was studied by using different PCR-based fingerprinting methods. Characteristic DNA profiles for 20 B. japonicum field isolates and two reference strains were obtained using random primers (RAPD) and two sets of repetitive primers (REP- and ERIC-PCR). In comparison with the REP, the ERIC primer set generates fingerprints of lower complexity, but still several strain-specific bands were detected. Different B. japonicum isolates could be more efficiently distinguished by using combined results from REP- and ERIC-PCR. The most polymorphic bands were observed after amplification with four different RAPD primers. Both methods, RAPD and rep-PCR, resulted in identical grouping of the strains. Cluster analysis, irrespective of the fingerprinting method used, revealed that all the isolates could be divided into three major groups. Within the major groups, the degree of relative similarity between B. japonicum isolates was dependent upon the method used. Our results indicate that both RAPD and rep-PCR fingerprinting can effectively distinguish different B. japonicum strains. RAPD fingerprinting proved to be slightly more discriminatory than rep-PCR.