Diversity of fungal latent pathogens and true endophytes associated with fruit trees in Uruguay

We have explored the fungal diversity in asymptomatic twigs of apple, peach, pear and blueberry trees, with the objective of discerning between true endophytes and latent pathogens. Several fungal genera containing known bark pathogens were found. Seven Diaporthe species—D. oxe, D. infecunda, D. serafiniae, D. phaseolorum, D. terebinthifolii, D. foeniculina and D. brasiliensis—were identified, along with Botryosphaeria dothidea, Neofusicoccum parvum, Neofusicoccum australe, Cytospora sp., Cytospora acaciae and Pestalotiopsis spp. A pathogenicity trial was undertaken to determine the role of these species on apple, pear, blueberry and peach shoots. Diaporthe brasiliensis, D. foeniculina, Diaporthe inconspicua, D. terebinthifolii, Diaporthe sp.1, Cytospora‐like isolates and Pestalotiopsis spp. isolates produced no lesions on inoculated shoots, suggesting that they could be considered true endophytes on their respective hosts. Meanwhile, some of the isolates of Diaporthe—D. oxe, Diaporthe sp.2, D. infecunda and D. serafiniae, B. dothidea, N. parvum and N. australe could be regarded as latent pathogens in their respective hosts as they produced sunken cankers and necrosis on inoculated shoots. These results demonstrate that apple, pear, blueberry and peach healthy shoots can host many known endophytic fungi along with potential wood disease‐causing fungi that should be regarded as latent pathogens.     

Neofusicoccum parvum and Diaporthe foeniculina associated with twig and shoot blight and branch canker of citrus in Greece

In a survey performed in Chania and Aetoloacarnania, Greece in years 2013–2014, fungal isolates causing twig and shoot blight and branch canker of citrus trees were morphologically characterized and identified by multiple gene sequence analysis. By sequencing the ITS‐5.8S rRNA, the elongation factor 1‐α (EF1‐α), the β‐tubulin and the RNA polymerase II subunit (Rpb2) genes, the isolates examined were associated with Diaporthe foeniculina (six isolates) and Neofusicoccum parvum (one isolate). All six D. foeniculina isolates showed slow colony growth rates (7.4 ± 3.2 mm/day), while the N. parvum isolate exhibited fast growth (41.6 mm/day). Koch's criteria were met after re‐isolation of D. foeniculina isolates from all inoculated Citrus spp. and N. parvum from inoculated C. reticulata “Ortanique” and after having developed symptoms similar to those detected on shoots and branches collected from citrus fields. Based on lesion length on detached C. medica “Lia Kritis” shoots, N. parvum caused long necrotic lesions (58 mm in length) in comparison with a length of 12–21 mm lesions caused by D. foeniculina isolates. Pathogenicity trials on nine Citrus spp., which had been inoculated with D. foeniculina and N. parvum, revealed different levels of susceptibility, indicating a host‐dependent infection effect, with Poncirus trifoliate × C. paradisi (“Citrumelo Swingle”) being the most resistant citrus genotype. Lack of host specificity suggests that their pathogen–host association could be attributed to ecological rather to co‐evolutionary factors. This work represents the first report, accompanied with pathogenicity tests, on botryosphaeriaceous and diaporthaceous pathogens associated with twig and shoot blight and branch canker of citrus in Greece.     

Botryosphaeriaceae associated with <Emphasis Type="Italic">Terminalia catappa</Emphasis> in Cameroon,South Africa and Madagascar

Species in the Botryosphaeriaceae represent some of the most important fungal pathogens of woody plants. Although these fungi have been relatively well studied on economically important crops, hardly anything is known regarding their taxonomy or ecology on native or non-commercial tree species. The aim of this study was to compare the diversity and distribution of the Botryosphaeriaceae on Terminalia catappa, a tropical tree of Asian origin planted as an ornamental in Cameroon, Madagascar and South Africa. A total of 83 trees were sampled, yielding 79 Botryosphaeriaceae isolates. Isolates were initially grouped based on morphology of cultures and conidia. Representatives of the different morphological groups were then further characterised using sequence data for the ITS, tef 1-alpha, rpb2, BOTF15 and beta-tub gene regions. Five species of the Botryosphaeriaceae were identified, including Neofusicoccum parvum, N. batangarum sp. nov., Lasiodiplodia pseudotheobromae, L. theobromae and L. mahajangana sp. nov. Lasiodiplodia pseudotheobromae and L. theobromae, were the most commonly isolated species (62%), and were found at all the sites. Neofusicoccum parvum and N. batangarum were found in South Africa and Cameroon, respectively, whereas L. mahajangana was found only in Madagascar. Greenhouse inoculation trials performed on young T. catappa trees showed variation among isolates tested, with L. pseudotheobromae being the most pathogenic. The Botryosphaeriaceae infecting T. catappa appear to be dominated by generalist species that also occur on various other hosts in tropical and sub-tropical climates.     

Molecular investigation of carbapenem resistance among multidrug‐resistant Pseudomonas aeruginosa isolated clinically in Thailand

Carbapenem resistant Pseudomonas aeruginosa were isolated among multidrug‐resistant (CR‐MDR) organisms from tertiary hospitals in Thailand. Decreased expression of oprD mRNA (93.65%) was predominant followed by increased expression of mexAB‐oprM mRNA (92.06%) and mexXY mRNA (63.49%). Interestingly, 23 of 126 (18.25%) isolates were susceptible to imipenem with down‐regulated oprD expression and non‐up‐regulated mexCD‐oprJ mRNA expression. Metallo‐β‐lactamases production was clearly positive in 24 isolates (18.46%) and weakly positive in 12 isolates (9.23%). Among both of these sets of isolates, imp‐1, imp‐14 and vim‐2 were identified. Hyperproduction of AmpC β‐lactamase had the lowest prevalence rate (3.97%). It was concluded that CR‐MDR P. aeruginosa clinical isolates in Thailand possess multifactorial resistance mechanisms.     

The prevalence of plasmid‐mediated quinolone resistance determinants among clinical isolates of ESBL or AmpC‐producing Escherichia coli from Chinese pediatric patients

Three kinds of PMQR determinants (qnr genes, aac(6’)‐Ib‐cr, and qepA) have been discovered and shown to be widely distributed among clinical isolates. To characterize the prevalence of PMQR determinants in ESBL or AmpC‐producing E. coli clinical isolates in Chinese children, a total of 292 ESBL or AmpC‐producing E. coli clinical isolates collected from five children's hospitals in China from 2005 to 2006 were screened for PMQR determinants by PCR. Twenty (6.8%) of the 292 isolates were positive for PMQR determinants. A total of 12 (4.1%) isolates were positive for qnr genes, comprising three positive for qnrA (1.0%), three for qnrB (1.0%), and six for qnrS (2.1%). Twenty‐four (8.2%) isolates were positive for aac(6’)‐Ib, of which 10 (3.4% of 292) had the –cr variant. There was no qepA gene detected in the isolates. Conjugation revealed that qnr, aac(6’)‐Ib‐cr, and ESBL‐encoding genes were transferred together.     

Does Hyphal‐Tip Ensure the Same Allelic Composition at SSR Loci as Monosporic Isolates of Sclerotinia sclerotiorum?

The proper characterization of individual is a basic stage in population genetic studies. In Sclerotinia sclerotiorum, genetic uniformity of an individual can be obtained by isolation of single ascospore; however, hyphal‐tip isolates are commonly used in genetic studies. The aim of this study was to assess whether hyphal‐tip isolates of S. sclerotiorum can be used as surrogate of monoascosporic (monosporic) isolates. Twenty‐eight isolates of S. sclerotiorum were collected from common bean plants with white mold symptoms and were purified by hyphal‐tip or single ascospore. The correspondence between hyphal‐tip and monosporic isolates was assessed through the allelic composition at 10 microsatellite (SSR) loci of the isolates obtained by both methods. For the SSR loci comprised of dinucleotide repeats in 92% of the cases, the difference (di) between the amplicon size values for hyphal‐tip and monosporic isolates was no more than one base pair. For the loci comprised of tetra or pentanucleotide repeats in 89% of the cases, di was no more than one base pair. The same allelic profile was found for hyphal‐tip or single ascospore isolates of S. sclerotiorum. When monosporic isolates cannot be easily obtained, hyphal‐tip can safeguard the genotypic identity of S. sclerotiorum isolates.     

Dissemination of ceftazidime-resistant Acinetobacter baumannii clonal complex 92 in Korea

Aims: This study was performed to describe the epidemiological traits of ceftazidime‐resistant Acinetobacter baumannii clinical isolates from Korea. Methods and Results: Antimicrobial susceptibilities were determined by disk diffusion assay. PCR experiments were performed to detect genes encoding extended‐spectrum β‐lactamases and metallo‐β‐lactamases. Detection of ISAba1 upstream of the bla_ADC gene was also performed by PCR amplification. The genetic organization of the bla_PER‐1 gene was investigated by PCR mapping and sequencing of the regions surrounding the gene. Multilocus sequence typing was performed using seven housekeeping genes. A. baumannii isolates of clonal complex (CC) 92 exhibited a higher resistance rate (286/289, 99%) against ceftazidime compared to A. baumannii isolates of non‐CC92 (7/87, 8%). Amongst 286 ceftazidime‐resistant isolates of CC92, 100 (35%) isolates carried the bla_PER‐1 gene, while none of the 87 isolates of non‐CC92 carried the gene. The bla_ADC gene associated with an ISAba1 element was detected in 98% (281/286) of ceftazidime‐resistant isolates of CC92 and in all seven ceftazidime‐resistant isolates of non‐CC92. The bla_PER‐1 gene was located on a transposon, Tn1213 (ISPa12‐bla_PER‐1‐Δgst‐ISPa13), in 95 isolates and on a complex class 1 integron (orf513‐bla_PER‐1‐putative ABC transporter gene) in five isolates. Southern blot experiments confirmed the chromosomal location of the bla_PER‐1 gene. Conclusions: Acinetobacter baumannii CC92 which has acquired ceftazidime resistance by the production of PER‐1 extended‐spectrum β‐lactamases and/or the overproduction of Acinetobacter‐derived cephalosporinase is widely disseminated in Korea. Significance and Impact of the Study: This study shows the mechanisms of acquiring ceftazidime resistance in A. baumannii and the epidemiological traits of ceftazidime‐resistant A. baumannii isolates from Korea.     

Vegetative Compatibility,Pathogenicity and Virulence Diversity of Fusarium oxysporum f. sp. melongenae Recovered from Eggplant

Fusarium oxysporum (Schlechtend.: Fr.) f. sp. melongenae (Fomg) recovered from symptomatic eggplants from five eggplant‐growing areas in Turkey, including the south, west, north‐west, north and south‐east regions. The objective of this study was to investigate the genetic diversity of the Fomg isolates from different geographical location by pathogenicity and VCG tests. Three hundred and seventy‐four Fomg isolates were classified as highly virulent, virulent, moderately virulent and low virulent through pathogenicity assays. No correlation was observed between virulence of Fomg isolates and their locations. The nitrate non‐utilizing mutants (nit) were generated as nit1, nit3 and NitM, based on phenotyping of Fomg growth characteristics of the Fomg isolates on diagnostic media with various sources of nitrogen. The majority of nit mutants (39.4%) recovered were nit1 from minimal medium (MM) containing of 2.0% potassium chlorate (MMC). The most of Fomg isolates were identified as heterokaryon self‐compatible (HSC) based on their ability to form a stable heterokaryon, while four isolates were classified as heterokaryon self‐incompatible (HSI). A large amount of Fomg isolates were vegetatively compatible and assigned as members of the same VCG, whereas nit mutants of 10 Fomg isolates that did not complement with tester strains only paired by themselves (HSC), these isolates were termed vegetative incompatible (vic). The complementation of 33 isolates with tester strains was slow and quite weak, but not paired with themselves even though they are HSC. About 96.3% of the Fomg isolates were assigned to VCG 0320, while the remaining 3.7% were classified as vegetative incompatible group.     

MORPHOLOGICAL AND PHYLOGENETIC STUDIES ON UNICELLULAR DIAZOTROPHIC CYANOBACTERIA (CYANOPHYTES) ISOLATED FROM THE COASTAL WATERS AROUND SINGAPORE1

Six unicellular diazotrophic cyanobacteria were isolated from the coast around Singapore. The isolates grew under both light:dark (L:D) cycles and continuous illumination (CL) in media without combined nitrogen and exhibited an ability to fix nitrogen (as measured by acetylene reduction) under aerobic conditions. The cells of all isolates were surrounded by a thick fibrous outer wall layer, and they divided by transverse binary fission. The arrangement of photosynthetic thylakoids was of the dispersed type. Three isolates were identified as form‐genus Gloeothece as cells were divided in a single plane, and the other three isolates were identified as form‐genus Gloeocapsa as cells were divided in multiple planes. Phylogenetic analyses based on the DNA sequences of the genes encoding 16S rRNA and dinitrogenase reductase (nifH) revealed the following: (i) Our six isolates formed a monophyletic cluster. (ii) The monophyletic cluster was subdivided into two phylogenetic groups, which taxonomically corresponded with the form‐genera Gloeothece and Gloeocapsa. However, (iii) a diazotrophic strain of form‐genus Gloeothece, Gloeothece membranacea (Rabenh.) Bornet PCC6501, was not closely related to our isolates, and (iv) some, but not all, diazotrophic unicellular strains of form‐genus Cyanothece were observed to be in a close relationship with our isolates.     

Biological properties of necrotic and non-necrotic strains of bean yellow mosaic virus in cool season grain legumes

A collection of 51 bean yellow mosaic virus (BYMV) isolates was transmitted from infected Trifolium subterraneum (subterranean clover) to Lupinus angustifolius (narrow‐leafed lupin) by Myzus persicae (green peach aphid). Depending on isolate and L. angiistifolius genotype used, two distinct responses developed in L. angustifolius plants, either systemic necrosis and plant death or non‐necrotic reactions of varying severity. Ten isolates caused necrosis and plant death in cv. Danja. However, when nine of these were inoculated to breeding line 90L423‐07‐13, seven induced non‐necrotic reactions, while two caused necrosis and plant death. Thirty seven isolates always produced non‐necrotic reactions regardless of genotype of L. angustifolius inoculated. Non‐necrotic and necrotic isolates originally came both from lupins and other species, and the non‐necrotic isolates were no less efficiently transmitted by M. persicae than the necrotic ones. When one isolate of each type was inoculated together to T. subterraneum and nine months later this culture was used as an acquisition source for aphid transmission to L. angustifolius, only the necrotic type was detected. Previous infection of L. angustifolius plants with a non necrolic isolate prevented subsequent infection by a necrotic one. All necrotic and non‐necrotic isolates reacted with BYMV antiserum in ELISA but only two cross‐reacted with antiserum to clover yellow vein virus (CYVV). When selected necrotic and non‐necrotic isolates were inoculated to differential hosts, all behaved like BYMV and not CYVV. When three isolates of each type were transmitted to 11 other cool season grain legume species, except in Cicer arietinum (chickpea), there were no necrotic reactions, but symptom severity varied with the isolate and species inoculated. The two isolates that caused necrosis in C. arietinum did not do so in L. angustifolius. The six isolates from Vicia faba (faba bean) all caused non‐necrotic reactions in L. angustifolius cv. Danja and 90L423‐07‐13. These and two necrotic isolates readily infected five genotypes of V. faba always causing severe symptoms. However, three non‐necrotic isolates from L. angustifolius and a further necrotic isolate were poorly infectious on V. faba in which they generally induced mild symptoms. These results show that at least three strain groups of BYMV can be distinguished by their reactions in different L. angustifolius genotypes, one causing necrosis and death in cv. Danja and 90L423‐07‐13, one causing necrosis and death in Danja but not 90L423‐07‐13, and one causing non‐nccrotic reactions in both. These strain groups could not be distinguished when representative necrotic and non‐necrotic isolates were inoculated to other grain legume species. However, inoculation to V. faba distinguished two other BYMV strain groupings differing in severity of symptoms and ability to infect this species.     

Occurrence and molecular characteristics of antimicrobial resistance of Escherichia coli from broilers,pigs and meat products in Thailand and Cambodia provinces

Nine hundred and forty‐one samples were collected in Sa Keao, Thailand (n = 554) and Banteay Meanchey, Cambodia (n = 387) from July 2014 to January 2015. A total of 667 Escherichia coli isolates (381 isolates from Sa Keao and 286 isolates from Banteay Meanchey) were obtained and examined for antimicrobial susceptibility, class 1 integrons, ESBL genes and horizontal transfer of resistance determinants. Prevalence of E. coli in pig and broiler carcass samples from slaughterhouses and fresh markets was 36–85% in Sa Keao and 11–69% in Banteay Meanchey. The majority of these isolates were multidrug resistant (75.3%). Class 1 integrons were common in both Thai (47%) and Cambodian (62%) isolates, of which four resistance gene cassette arrays including aadA1, dfrA1‐aadA1, dfrA12‐aadA2 and aadA2‐linF were identified. Class 1 integrons in two broiler isolates from Sa Keao (dfrA12‐aadA2) and one broiler isolate from Banteay Meanchey (dfrA1‐aadA1) were horizontally transferable. Sixteen isolates were confirmed to be ESBL‐producing strains with ESBL gene bla_CTX‐M‐15, broad spectrum β‐lactamase gene bla_TEM‐1 and the AmpC gene bla_CMY‐2 being detected. The bla_TEM‐1 gene was most prevalent and located on a conjugative plasmid.     

Antibiotic-resistant Salmonella and Escherichia coli isolates with integrons and extended-spectrum beta-lactamases in surface water and sympatric black-headed gulls

Aim: To examine surface water from a pond in the northeastern part of the Czech Republic and young black‐headed gulls (Larus ridibundus) nesting on the same pond for the presence of antibiotic‐resistant Salmonella and Escherichia coli. Methods and Results: A total of 16% (n = 87) of water and 24% (n = 216) of gull samples yielded Salmonella. Salmonella Enteritidis PT8 and PT4 were the most prevalent. Antibiotic resistance was found in 12% (n = 14) of water and 28% (n = 51) of gull salmonellae. Escherichia coli were found in 83 (95%) and 213 (99%) of pond water and gull samples, respectively. Totals of 18% (n = 83) of water and 28% (n = 213) of gull E. coli isolates were resistant to antimicrobial agents tested. Class 1 integrons were found in 21% (n = 14) of water and 15% (n = 60) of gull antibiotic‐resistant E. coli isolates. Class 2 integrons and extended‐spectrum beta‐lactamase‐producing E. coli isolates (with bla_CTX‐M‐1, bla_CTX‐M‐15‐like, bla_SHV‐2 and bla_SHV‐12) were found in 13% (eight positive, n = 60 gull‐resistant E. coli isolates) and 3% (seven positive, n = 216 gull E. coli isolates) of gull isolates, respectively. Antibiotic‐resistant E. coli isolates with identical pulsed field gel electrophoresis (PFGE) patterns were found in either gulls or water, but not both. Salmonellae of the same serotype and PFGE profile were found in both gulls and water. Conclusion: A high prevalence of antibiotic‐resistant salmonellae and E. coli were found in both pond water and in sympatric black‐headed gulls. Significance and Impact of the Study: Intensive contamination of pond surface water by antibiotic‐resistant E. coli and salmonellae was documented. Black‐headed gulls were identified as important reservoirs of antibiotic‐resistant salmonellae and E. coli, including extended‐spectrum beta‐lactamase‐producing isolates.     

Horticultural mineral oil treatments in nurseries during aphid flights reduce Plum pox virus incidence under different ecological conditions

The application of horticultural mineral oil (HMO) treatments has been reported as a possible control strategy to reduce Plum pox virus (PPV) incidence in Prunus nurseries. The effect of Sunspray Ultrafine HMO at 1% on the natural viral spread was evaluated in experimental nursery plots of Nemaguard and Mariana GF8‐1 Prunus rootstock blocks established under high natural inoculum pressure of the most prevalent PPV‐types. Tests were conducted in experimental nursery plots in Plovdiv, Bulgaria (PPV‐M and PPV‐Rec), in Bistrita, Romania (PPV‐D and PPV‐Rec) and in Llíria, Spain (PPV‐D). Horticultural mineral oil treatments were applied weekly during the vegetative period from spring to fall (treatments were interrupted in the summer). Nursery plants were analysed yearly by double‐antibody sandwich enzyme‐linked immunosorbent assay with 5B‐IVIA/AMR monoclonal antibodies. The population dynamics of the aphids visiting plants in each experimental nursery plot was monitored by the sticky‐shoot method and also by Moericke yellow water traps. At all three locations, the aphid population first peaked in the springtime. Furthermore, a variable second peak of aphid population was observed in Plovdiv and Bistrita in autumn. The treatments reduced PPV incidence in the three experimental locations and plots and in both assayed Prunus rootstocks grown under high PPV‐inoculum pressure. A reduction from 10% to 20% of PPV‐incidence between treated and control plants (P < 0.05) in Plovdiv and Bistrita, respectively, was observed at the end of the tests. However, HMO treatments did not prevent PPV infection altogether, probably because of the high PPV prevalence in the area near the experimental nursery blocks. The control of PPV in nursery blocks based on HMO is presented as an environmentally friendly strategy based on the physical action of the treatments.     

Emergence of carbapenem non‐susceptible multidrug resistant Acinetobacter baumannii strains of clonal complexes 103B and 92B harboring OXA‐type carbapenemases and metallo‐β‐lactamases in Southern India

The molecular epidemiology and carbapenem resistance mechanisms of clinical isolates of Acinetobacter baumannii obtained from a south Indian tertiary care hospital were investigated by repetitive extragenic palindromic sequence PCR (REP‐PCR) and multi‐locus sequence typing (MLST). Analysis of resistant determinants was achieved by PCR screening for the presence of genes encoding OXA‐carbapenemases, metallo‐β‐lactamases (MBLs) and efflux pumps. REP‐PCR generated around eight clusters of high heterogeneity; of these, two major clusters (I and V) appeared to be clonal in origin. Analysis of representative isolates from different clusters by MLST revealed that most of the isolates belonged to sequence type 103 of CC103^B. Second most prevalent ST belonged to clonal complex (CC) 92^B which is also referred to as international clone II. Most of the isolates were multi‐drug resistant, being susceptible only to polymyxin‐B and newer quinolones. Class D β‐lactamases such as bla_{OXA‐51‐like} (100%), bla_{OXA‐23‐like} (56.8%) and bla_{OXA‐24‐like} (14.8%) were found to be predominant, followed by a class B β‐lactamase, namely bla_IMP‐1 (40.7%); none of the isolates had bla_OXA‐58 like, bla_NDM‐1 or bla_SIM‐1. Genes of efflux‐pump adeABC were predominant, most of isolates being biofilm producers that were PCR‐positive for autoinducer synthase gene (>94%). Carbapenem non‐susceptible isolates were highly diverse and present throughout the hospital irrespective of type of ward or intensive care unit. Although previous reports have documented diverse resistant mechanisms in A. baumannii, production of MBL and OXA‐type of carbapenamases were found to be the predominant mechanism(s) of carbapenem resistance identified in strains isolated from Southern India.     

Occurrence and Genome Analysis of Cucurbit chlorotic yellows virus in Iran

In 2011 and 2012, several cucurbit‐growing regions of Iran were surveyed and samples with symptoms similar to those induced by Cucurbit chlorotic yellows virus (CCYV) were collected. The pathogen was transmitted to cucumber and melon under greenhouse conditions by whiteflies (Bemisia tabaci). RT‐PCR using designed CCYV‐specific primer pair (CCYV‐F/CCYV‐R) resulted in amplification of the predicted size DNA fragment (870 bp) for the coat protein (CP) gene in samples collected from Boushehr, Eyvanakay and Varamin. Nucleotide sequences of the CP of the three Iranian CCYV isolates were compared with five CCYV isolates obtained from GenBank and analysed. Phylogenetically, all CCYV isolates clustered in two groups; Group I is composed of five non‐Iranian isolates from China, Lebanon, Japan, Sudan and Taiwan, and the three Iranian isolates formed Group 2. Among Iranian isolates, the Eyvanakay isolate clustered in a distinct clade with the Boushehr and Varamin isolates. A phylogenetic tree based on amino acid identity of CP showed that CCYV was closely related to Lettuce chlorosis virus (LCV), Bean yellow disorder virus (BnYDV) and Cucurbit yellow stunting disorder virus (CYSDV). This is the first report of CCYV in Iran.     

Morphological, Pathological and Molecular Variability in Colletotrichum capsici, the Cause of Fruit Rot of Chillies in the Subtropical Region of North-western India

Fruit rot of chillies (Capsicum annuum L.), caused by Colletotrichum capsici under tropical and subtropical conditions, results in qualitative and quantitative yield losses. Based on variation in cultural and morphological traits of C. capsici populations, 37 isolates were categorized into five groups designated, respectively, as Cc‐I, Cc‐II, Cc‐III, Cc‐IV and Cc‐V. In culture, most of the isolates produced cottony, fluffy or suppressed colonies. However, no significant differences were noticed in shape and size of conidia. The reaction of the 37 isolates on an indigenously developed differential set of Capsicum cultivars indicated the existence of different virulences in Himachal Pradesh (HP) chilli populations. Fifteen pathotypes of the pathogen were characterized from various chilli‐growing regions of HP. Pathotype CCP‐1 was most virulent and attacked all the differential cultivars. The genetic relationship between five morphological groups recognized within C. capsici was investigated using random amplified polymorphic DNA (RAPD) analysis. Molecular polymorphism generated by RAPD confirmed the variation in virulences of C. capsici and different isolates were grouped into five clusters. However, four isolates (Cc‐5, Cc‐33, Cc‐29 and Cc‐37) exhibited identical RAPD haplotypes. The pathological and RAPD grouping of isolates suggested no correlation among the test isolates.     

Genetic Diversity and Pathogenicity of Fusarium oxysporum f.sp. melonis Races from Different Areas of Italy

Fusarium wilt caused by Fusarium oxysporum f.sp. melonis (FOM) is a devastating disease of melon worldwide. Pathogenicity tests performed with F. oxysporum isolates obtained from Italian melon‐growing areas allowed to identify thirty‐four FOM isolates and the presence of all four races. The aims of this work were to examine genetic relatedness among FOM isolates by race determination and to perform phylogenetic analyses of identified FOM races including also other formae speciales of F. oxysporum of cucurbits. Results showed that FOM race 1,2 was the most numerous with a total of eighteen isolates, while six and nine isolates were identified as race 0 and 1, respectively, and just one isolate was assigned to race 2. Phylogenetic analysis was performed by random amplified polymorphic DNA (RAPD) profiling and by translation elongation factor‐1α (TEF‐1α) sequencing. The analysis of RAPD profiles separated FOM races into two distinct clades. Clade 1, which included races 0, 1 and 1,2, was further divided into ‘subclade a’ which grouped almost all race 1,2 isolates, and into ‘subclade b’ which included race 0 and 1 isolates. Clade 2 comprised only race 2 isolates. The phylogenetic analysis based on TEF‐1α separated FOM from the other formae speciales of F. oxysporum. Also with TEF‐1α analysis, FOM races 0, 1 and 1,2 isolates grouped in one single clade clearly separated from FOM race 2 isolates which grouped closer to F. oxysporum f.sp. cucumerinum. RAPD technique was more effective than TEF‐1α in differentiating FOM race 1,2 isolates from those belonging to the closely related races 0 and 1. Both phylogenetic analyses supported the close relationship between the three different FOM races which might imply the derivation from one another and the different origin of FOM race 2.     

Rapid Detection and Identification of Six Tomato yellow leaf curl virus Isolates from Different Regions Using Polymerase Chain Reaction and Restriction Enzyme Analysis

The combinational analysis of polymerase chain reaction and restriction enzyme analysis (PCR‐RE) to distinguish six Tomato yellow leaf curl virus (TYLCV) isolates from five countries was developed. Tomato yellow leaf curl virus has spread from the Middle East to Western Europe, Central America and Eastern Asia, and occurs on infected crops such as tomatoes, peppers, cucurbits and beans. Tomato yellow leaf curl virus isolates from Jordan (TYLCV‐Mld[Jo:Cuc] and TYLCV‐IL[Jo:Cuc]), Israel (TYLCV‐IL[IL:Reo:86]), Spain (TYLCV‐Mld[ES72/97]), USA (TYLCV‐IL[US:F10:04]) and Korea (TYLCV‐KR) were collected, and the sequences of the six isolates were analysed to distinguish them by PCR‐RE combination analysis. Oligonucleotide primers for the six TYLCV isolates were designed to amplify approximately 740 base pairs including the intergenic region (IR) and parts of V1 and V2 ORF. Unique restriction enzyme sites were analysed to identify isolate‐specific restriction enzyme sites on the PCR products of each isolate. Three enzymes (DdeI, FauI and BssSI) were selected by in silico analysis, and then, the PCR products following the serial digestion of each restriction enzyme were separated by agarose gel electrophoresis to distinguish the TYLCV isolates. Taken together, the PCR‐RE combination analysis by serial digestion with three restriction enzymes could be a useful method for distinguishing the six isolates.