Morphological and molecular analysis of genetic variability within isolates of <Emphasis Type="Italic">Corynespora cassiicola</Emphasis> from different hosts

Twenty-two isolates of Corynespora cassiicola obtained from cucumber, papaya, eggplant, tomato, bean, Vigna, sesame and Hevea rubber (Hevea brasiliensis) were analysed by morphological features, the differences of the ribosomal DNA internal transcribed spacer (rDNA-ITS) region sequence and the inter simple sequence repeat (ISSR) technique. Variability of morphological features was observed among the isolates. Pathogenicity tests showed that isolates from different hosts attacked Hevea rubber. Sequences of two outgroup taxa, C. proliferata and C. citricola, were downloaded from GenBank. The phylogenetic trees were constructed by using the rDNA-ITS region sequences from 24 Corynespora spp. isolates. In this analysis, the 24 sequences grouped into two clusters (A and B). Cluster A consists of sequences from all isolates of C. cassiicola; whereas cluster B consists of the two outgroup taxa, C. proliferata and C. citricola. However, the ITS region is conservative, and is not fit for studying differences among isolates. A total of 114 DNA fragments was amplified with 16 ISSR primers, among which 102 were polymorphic (89.5%). A dendrogram was created by the unweighted pair-group method with arithmetic averaging (UPGMA) analysis, and 22 isolates grouped into three clusters (C, D and E). Cluster C is composed of all of the Hevea rubber isolates, whereas cluster D is composed of nine isolates: four from papaya, five from cucumber, eggplant, bean, vigna and sesame. Cluster E is composed of two isolates from cucumber and tomato. These analyses showed that the genetic diversity was very rich among the tested isolates. There are no correlations between the morphological characteristics or rDNA-ITS region sequences of the 22 isolates and their host or geographical origin, but there is a link between ISSR clusters and their host origins. ISSR markers appear to be useful for intra-species population study in C. cassiicola.     

Molecular diversity of binucleate <Emphasis Type="Italic">Rhizoctonia</Emphasis> AG-A in China

AG-A belongs to the binucleate Rhizoctonia (BNR) anastomosis group (AG) of the Ceratobasidium teleomorph, which parasitizes the roots of many plant species. Ninety nine isolate species of AG-A were obtained from Tibet, Sichuan, and Yunnan Province in China. All isolates were divided into three types based on their cultural characteristics. Type I: abundant aerial mycelia, dense hyphae, loose sclerotia; Type II: abundant aerial mycelia, no sclerotia. Type III: sparse aerial mycelium and no sclerotia. All of the isolates infected the seedlings of Chinese mustard and Chinese cabbage, causing the formation of lesions on the stem and a brown discoloration of the roots. Sequence analysis of the 5.8S rDNA-ITS showed a similarity of 98–100% among the isolates. Inter Simple Sequence Repeat (ISSR) was used to detect genetic variation in binucleate Rhizoctonia spp. Forty two AG-A isolates were amplified using 15 random primers. From a total of 164 bands, 144 bands (87.8%) were polymorphic in the 42 tested isolates. A dendrogram showing genetic relationships between the isolates was constructed using unweighted pair-group averages based on genetic distances. According to the dendrogram, the 42 tested isolates could be aligned into three clusters with a genetic similarity coefficient of 0.29, the first clusters including 27 isolates with III of culture characteristics on PDA; the second clusters included eight isolates with I of cultural characteristics on PDA; the third cluster included seven isolates with II of cultural characteristics on PDA. The results of ISSR analysis showed an association between the hosts of these isolates. Our results showed that ISSR analysis can reveal more molecular variation among isolates of AG-A than sequence analysis using the 5.8S rDNA-ITS.     

<Emphasis Type="Italic">Colletotrichum destructivum from</Emphasis> cowpea infecting <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and its identity to <Emphasis Type="Italic">C. higginsianum</Emphasis>

Colletotrichum isolates isolated from cowpea in the Hangzhou area of China were identified as C. destructivum based on morphological characteristics, pathogenicity tests, sequence analysis of the internal transcribed spacer (ITS)1, 5.8S RNA gene and ITS2 regions of ribosomal DNA and the infection process. The ability of the C. destructivum isolates to infect Arabidopsis thaliana was investigated under laboratory conditions and showed a two-phase hemibiotrophic infection process. In addition, the sequences of the rDNA ITS region of C. destructivum isolates from cowpea were identical with 100% similarity to that of isolates of C. higginsianum originating from cruciferous plants. This article presents new evidence in support of C. higginsianum as a synonym of C. destructivum.     

Multigene phylogenetic analysis of inter- and intraspecific relationships in <Emphasis Type="Italic">Venturia nashicola</Emphasis> and <Emphasis Type="Italic">V. pirina</Emphasis>

Isolates of Venturia species isolated from pear in Japan, China, Taiwan and Israel were used in this study to analyze their molecular phylogenetic relationship. The nucleotides of rDNA-ITS, partial β-tubulin and elongation factor 1α genes were sequenced directly after PCR. Based on these sequence data two phylogenetic groups could be distinguished. Isolates collected from Asian pears such as Japanese and Chinese pears formed a distinct evolutionary lineage from those derived from European and Syrian pears. This result corroborated the early taxonomic separation of V. nashicola from V. pirina. In addition, trees from single-locus data sets and the combined data set showed that all isolates of V. nashicola were included in a monophyletic group and representative isolates of five pathological races originating from different locations and cultivars formed a single lineage. In contrast, two distinct evolutionary lineages were revealed in V. pirina and isolates of five races were scattered in two lineages. Israeli isolates of race 2 as well as two Japanese isolates of V. pirina formed a distinct lineage from other isolates of this species, while other Israeli isolates belonging to races 1, 3, 4, and 5 were closely related to each other and formed another lineage. It was indicated that the evolution of pathological races in V. nashicola might have occurred relatively recently as compared with V. pirina.     

Anthracnose of salt-wort (<Emphasis Type="Italic">Salsola komarovii</Emphasis>) caused by <Emphasis Type="Italic">Colletotrichum truncatum</Emphasis>

In July 2006, black rot was observed on the leaves of 4-leaf-stage seedlings of salt-wort (Salsola komarovii) in Yamagata Prefecture, Japan. We isolated two single-conidial isolates from the diseased leaves. Although colony appearance of the isolates was different from that of each other, both isolates were identified as Colletotrichum truncatum by morphology and molecular similarity. After inoculation of healthy salt-wort plants with the isolates, the isolates were reisolated from symptomatic plants. We thus propose a new disease, anthracnose of salt-wort.     

First report of blueberry red ringspot disease caused by <Emphasis Type="Italic">Blueberry</Emphasis><Emphasis Type="Italic">red</Emphasis><Emphasis Type="Italic">ringspot</Emphasis><Emphasis Type="Italic">virus</Emphasis> in Japan

Virus-like symptoms—red ringspots on stems and leaves, circular blotches or pale spots on fruit—were found on commercial highbush blueberry (Vaccinium corymbosum) cultivars Blueray, Weymouth, Duke and Sierra in Japan. In PCR testing, single DNA fragments were amplified from total nucleic acid samples of the diseased blueberry bushes using primers specific to Blueberry red ringspot virus (BRRV). Sequencing analysis of the amplified products revealed 95.7–97.7% nucleotide sequence identity with the BRRV genome. This paper is the first report of blueberry red ringspot disease caused by BRRV in Japan. The nucleotide sequence data reported in this paper are available in the GenBank/EMBL/DDBJ database as accessions AB469884 to AB469893 for BRRV isolates from Japan.     

Leaf spot disease of <Emphasis Type="Italic">Hyoscyamus muticus</Emphasis> (Egyptian henbane) caused by <Emphasis Type="Italic">Cladosporium herbarum</Emphasis>

Severe brown spots caused by Cladosporium herbarum appeared on the leaves of Hyoscyamus muticus (Egyptian henbane) grown in a greenhouse at Yamaguchi city, Japan, in the summer of 2008. Nucleotide sequence analysis of rDNA-ITS and 28S rDNA supported the morphological identification of the isolate, which caused the same disease symptoms after reinoculation of the host. This new disease was named “Cladosporium leaf spot of Egyptian henbane”.     

Reaction of <Emphasis Type="Italic">Leersia</Emphasis> grasses to <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis> collected from Japan and Asian countries

The present study was conducted to determine if there is specificity in the host-pathogen relationship between the isolates of Xanthomonas oryzae pv. oryzae, the causal bacterium for rice blight and Leersia grasses, the alternative weed hosts of the disease. Plants of three species of Leersia, namely, L. sayanuka, L. oryzoides and L. japonica, were collected from various parts of Japan and were inoculated with the X. oryzae pv. oryzae isolates obtained from various locations in Japan and from 11 Asian countries. Four L. sayanuka plants were found susceptible to all Race II isolates and some Race I isolates, but were resistant to all Race III isolates. Race III is known to have a wider range pathogenicity to rice cultivar groups compared with Race I and II. Although the reactions of two L. oryzoides plants to Race I and II isolates were similar to that of L. sayanuka, the L. oryzoides plant collected from Niigata Prefecture showed a susceptible reaction to some Race III isolates. On the other hand, L. japonica plants gave reactions different those of L. sayanuka and L. oryzoides, with two plants of L. japonica found to be resistant to all test isolates collected from Japan. The Asian isolates exhibited a wide host range against the international differential rice cultivars, but almost all of them were avirulent to Leersia plants. These results indicate that the relationship between the pathogenicity of the causal bacterium and the resistance of host plants is very complex, and suggest that pathogenic diversity of X. oryzae pv. oryzae might be related to the resistance of Leersia spp.     

Pathogenicity,colony morphology and diversity of isolates of <Emphasis Type="Italic">Guignardia citricarpa</Emphasis> and <Emphasis Type="Italic">G. mangiferae</Emphasis> isolated from <Emphasis Type="Italic">Citrus</Emphasis> spp.

In the present study, the pathogenicity of 36 isolates of Guignardia species isolated from asymptomatic ‘Tahiti’ acid lime fruit peels and leaves, ‘Pêra-Rio’ sweet orange leaves and fruit peel lesions, and a banana leaf were characterized. For pathogenicity testing, discs of citrus leaves colonized by Phyllosticta citricarpa under controlled laboratory conditions were kept in contact with the peels of fruit that were in susceptible states. In addition, pathogenicity was related to morphological characteristics of colonies on oatmeal (OA) and potato dextrose agar (PDA). This allowed the morphological differentiation between G. citricarpa and G. mangiferae. Polymerase chain reactions (PCRs) were also used to identify non-pathogenic isolates based on primers specific to G. citricarpa. A total of 14 pathogenic isolates were detected during pathogenicity tests. Five of these were obtained from leaf and fruit tissues of the ‘Tahiti’, which until this time had been considered resistant to the pathogen. Given that the G. citricarpa obtained from this host was pathogenic, it would be more appropriate to use the term insensitive rather than resistant to categorize G. citricarpa. A non-pathogenic isolate was obtained from lesions characteristic of citrus black spot (CBS), indicating that isolation of Guignardia spp. under these conditions does not necessarily imply isolation of pathogenic strains. This also applied to Guignardia spp. isolates from asymptomatic citrus tissues. Using fluorescent amplified fragment length polymorphism (fAFLP) markers, typically pathogenic isolates were shown to be more closely related to one another than to the non-pathogenic forms, indicating that the non-pathogenic isolates display higher levels of genetic diversity.     

<Emphasis Type="Italic">Eremothecium coryli</Emphasis> and <Emphasis Type="Italic">E.</Emphasis><Emphasis Type="Italic">ashbyi</Emphasis> cause yeast spot of azuki bean

Yeast-like fungi were isolated from lesions on azuki bean (cv. Shin-Kyotodainagon) seeds that had been sucked by bean bugs in Kyoto Prefecture, Japan. On the basis of morphological and physiological characteristics and sequence data of the internal transcribed spacer (ITS) regions including the 5.8S rDNA, these yeasts were identified as Eremothecium coryli and E. ashbyi. Pathogenicity of those yeasts was confirmed by a reinoculation test. To our knowledge, this is the first report of the occurrence of yeast spot in azuki bean in Japan. The nucleotide sequence data reported are available in the GeneBank/EMBL/DDBJ database as accessions AB478291–AB478309 for E. coryli AZC1–19 and AB478310–AB478317 for E. ashbyi AZA1–8.     

Characterization of <Emphasis Type="Italic">Pectobacterium carotovorum</Emphasis> subsp. <Emphasis Type="Italic">carotovorum</Emphasis> causing soft-rot disease on <Emphasis Type="Italic">Pinellia ternata</Emphasis> in China

Pinellia ternata is a traditional Chinese herb which has been used in China for over 1,000 years. A soft-rot disease characterized by water-soaked lesions and soft-rot symptoms with a stinking odour was commonly observed in cultivated fields of this plant, and Pectobacterium-like bacteria were consistently isolated from the infected tissues. Two typical strains (SXR1 and ZJR1), isolated from Shanxi and Zhejiang, respectively, were identified. Pathogenicity tests revealed that these strains were virulent to P. ternata and induced the same symptoms as observed in the field. Characterization involving fatty acid profile, metabolic and physiological properties, 16S rDNA sequence and PCR-RFLP identified both isolates as P. carotovorum subsp. carotovorum (Pcc). The 16S rDNA of both isolates shared 97–99% sequence similarity with that of Pcc strains. The phylogenetic trees showed that both isolates were clustered in the group of Pcc and P. carotovorum subsp. odorifera and both PCR-RFLP profiles were consistent with the pattern E produced by the minority of Pcc strains. Thus, isolates SXR1 and ZJR1 were characterized as Pcc in spite of some differences. This is the first report that Pcc has been proven as a causal agent of soft-rot disease on P. ternata.     

Identification of <Emphasis Type="Italic">Pythium tracheiphilum</Emphasis> as the causal agent of vascular necrosis of endive (<Emphasis Type="Italic">Cichorium endivia</Emphasis>) in Spain

Pythium isolates were recovered from endive plants (Cichorium endivia) showing vascular necrosis collected from commercial fields located in Castellón province (eastern Spain). They were identified as Pythium tracheiphilum on the basis of their phenotypical and molecular profile. Pathogenicity tests conducted with two P. tracheiphilum isolates, obtained from endive and lettuce (Lactuca sativa), respectively, in this region, confirmed that both isolates were pathogenic to endive, with no significant differences in virulence between them. This is the first report of vascular necrosis caused by P. tracheiphilum on endive in Spain.     

<Emphasis Type="Italic">Cucurbit aphid-borne yellows virus</Emphasis> in Egypt

During 2010, yellowing symptoms were frequently observed in cultivated squash fields in Egypt. A total of 717 symptomatic squash leaf samples were collected from four regions where squash cultivation is of economic importance for the country: Kafrelsheikh, El-Behira, El-Sharkia and El-Ismailia. Serological analysis showed that 95.6% of the symptomatic squash samples were infected by Cucurbit aphid-borne yellows virus (CABYV), and visual estimation of the incidence of yellowing symptoms suggested a very high incidence of CABYV in the fields. Twelve CABYV isolates were characterized by sequencing two regions of the viral genome, open reading frame (ORF) 3 and ORFs 4/5. Overall, Egyptian isolates were very similar among them, and had higher similarity values with a French than with a Chinese isolate. The average nucleotide diversity for ORF 3 was significantly higher than for the other two regions, indicating that variability is not evenly distributed along the viral genome. The ratios between nucleotide diversity values in non-synonymous (d _N ) and synonymous (d _S) positions (d _N /d _S) for each ORF showed that the three ORFs are evolving under different pressures, although predominantly under purifying selection. Phylogenetic analyses revealed that these Egyptian isolates, with only one exception, shared the same clade with a French isolate. Moreover, these analyses suggested that Egyptian isolates belong to the Mediterranean group described previously.     

<Emphasis Type="Italic">Mycosphaerella</Emphasis> species occurring on <Emphasis Type="Italic">Eucalyptus globulus</Emphasis> in Portugal

Mycosphaerella leaf disease (MLD) is caused by species of Mycosphaerella and several anamorphic form genera that have been connected to Mycosphaerella. Until recently, MLD of eucalypts was largely ignored in Portugal. However, serious damage to Eucalyptus globulus has been reported since 1999 when frequent and severe defoliation of young trees was observed. The severity of this disease prompted a preliminary study of the Mycosphaerella species associated with major symptoms of a leaf blotch disease in commercial plantations of E. globulus in Portugal, which is presented here. The species were identified by molecular methods based on the ITS1-5.8S-ITS2 cluster, together with morphological characters. In addition to confirming the species previously recorded, Mycosphaerella vespa is reported for the first time from Portugal, while the status of Mycosphaerella grandis remains to be resolved.     

Fitness cost of virulence differs between the <Emphasis Type="Italic">AvrLm1</Emphasis> and <Emphasis Type="Italic">AvrLm4</Emphasis> loci in <Emphasis Type="Italic">Leptosphaeria maculans</Emphasis> (phoma stem canker of oilseed rape)

To investigate whether the reported fitness cost of virulence at the AvrLm4 locus in Leptosphaeria maculans is common to other loci, near-isogenic (NI) isolates differing at AvrLm1 locus were produced in vitro. Fitness of virulent (avrLm1) or avirulent (AvrLm1) isolates on Brassica napus without the corresponding R (resistance) gene Rlm1 was investigated in controlled environment (CE) and field experiments. Results indicate that there is a measurable fitness cost for avrLm1 compared to AvrLm1 isolates in terms of number of lesions, size of lesions, distance grown through leaf tissue towards the petiole in CE experiments and systemic growth from leaf lesions to stems in field experiments. There were differences in fitness cost between the AvrLm1 and AvrLm4 loci. There was a cultivar effect on fitness cost of virulence at the AvrLm1 locus but not at the AvrLm4 locus. In CE experiments, the optimal temperature for leaf infection was greater for AvrLm4 isolates than for AvrLm1 isolates. Field experiment results suggest that on the same host AvrLm4 isolates are more fit than AvrLm1 isolates in warmer seasons. The fitness cost at the AvrLm4 locus was generally greater than at the AvrLm1 locus, suggesting that the corresponding R gene Rlm4 may be more suitable than Rlm1 for redeployment in commercial cultivars after an interval of a few years.     

Identification and characterization of the <Emphasis Type="Italic">Enterobacter</Emphasis> complex causing mulberry (<Emphasis Type="Italic">Morus alba</Emphasis>) wilt disease in China

Mulberry wilt disease (MWD) was recently identified in Hangzhou, Zhejiang province, China. Typical symptoms of the disease are browning of vascular tissues, leaf wilt, defoliation, and tree decline. Unlike the symptoms of bacterial wilt disease caused by Ralstonia solanacearum, symptoms of MWD generally started from the bottom of the plants and moved upward. In inoculation experiments, four selected MWD strains caused mulberry shoot leaf wilt, discoloration, and defoliation. They also induced whole plant leaf wilt, defoliation and dark brown discoloration of vascular tissue. Based on Biolog metabolic profiles, fatty acid methyl ester analysis (FAME) and sequence analysis of the partial 16S rDNA and rpoB genes four MWD strains were identified as members of the genus Enterobacter. The 16S rDNA and rpoB gene sequences revealed a close relationship among two isolates, R2-2 and R6-2, and the E. asburiae type strain JCM6051. The isolates showed >98% similarity to E. asburiae JCM6051 in their rpoB gene. These results indicated that isolates R2-2 and R6-2 belonged to E. asburiae. No similarity in 16S rDNA sequences above 97% was found between either of the remaining isolates, R11-2 or R18-2, and any recognized Enterobacter species, suggesting that the two isolates may represent novel Enterobacter species. rpoB gene similarity values between the isolates and Enterobacter spp. type strains were <98%, providing further evidence that the two isolates may represent a novel species within the Enterobacter. The causal agent for MWD was previously reported to be E. cloacae, however, this study found that other Enterobacter spp. (E. asburiae and Enterobacter sp.) also cause MWD.     

Genetic variation among <Emphasis Type="Italic">Fusarium</Emphasis> isolates from onion,and resistance to <Emphasis Type="Italic">Fusarium</Emphasis> basal rot in related <Emphasis Type="Italic">Allium</Emphasis> species

The aim of this research was to study levels of resistance to Fusarium basal rot in onion cultivars and related Allium species, by using genetically different Fusarium isolates. In order to select genetically different isolates for disease testing, a collection of 61 Fusarium isolates, 43 of them from onion (Allium cepa), was analysed using amplified fragment length polymorphism (AFLP) markers. Onion isolates were collected in The Netherlands (15 isolates) and Uruguay (9 isolates), and received from other countries and fungal collections (19 isolates). From these isolates, 29 were identified as F. oxysporum, 10 as F. proliferatum, whereas the remaining four isolates belonged to F. avenaceum and F. culmorum. The taxonomic status of the species was confirmed by morphological examination, by DNA sequencing of the elongation factor 1-α gene, and by the use of species-specific primers for Fusarium oxysporum, F. proliferatum, and F. culmorum. Within F. oxysporum, isolates clustered in two clades suggesting different origins of F. oxysporum forms pathogenic to onion. These clades were present in each sampled region. Onion and six related Allium species were screened for resistance to Fusarium basal rot using one F. oxysporum isolate from each clade, and one F. proliferatum isolate. High levels of resistance to each isolate were found in Allium fistulosum and A. schoenoprasum accessions, whereas A. pskemense, A. roylei and A. galanthum showed intermediate levels of resistance. Among five A. cepa cultivars, ‘Rossa Savonese’ was also intermediately resistant. Regarding the current feasibility for introgression, A. fistulosum, A. roylei and A. galanthum were identified as potential sources for the transfer of resistance to Fusarium into onion.     

Immunodetection of the replicative complex of <Emphasis Type="Italic">Barley yellow dwarf</Emphasis><Emphasis Type="Italic">virus</Emphasis>-PAV <Emphasis Type="Italic">in vivo</Emphasis>

The genomic fragments of two open reading frames (ORFs) 1 and 2 of German and Canadian PAV isolates of Barley yellow dwarf virus (BYDV-PAV) were sequenced. Sequences only slightly differed from previously published sequences of this virus. Two polyclonal antisera against proteins encoded by ORFs 1 and 2 of a German ASL-1 isolate were developed using recombinant antigens expressed in E. coli as a fusion either to His₆− or thioredoxin-tags. In Western blot analysis with total protein extracts from BYDV infected plants, antisera efficiently recognized the 99 kDa fusion protein expressed from ORF1 and ORF2 (P1–P2 protein). Later in infection the P1–P2 protein disappeared and two smaller proteins, revealing sizes of 39 and 60 kDa, could be detected.     

Postharvest anthracnose on Satsuma mandarin orange caused by <Emphasis Type="Italic">Colletotrichum fioriniae</Emphasis>

In early January 2015–2017, anthracnose was detected on Satsuma mandarin orange (SMO) (Citrus unshiu) fruits kept in farmers’ storage rooms in Saga Prefecture, Japan. Three single-spore isolates from rotten fruits reproduced the postharvest anthracnose symptoms in wound-inoculated SMO fruits and were re-isolated from lesions. The isolates were identified as Colletotrichum fioriniae based on conidial morphology, culture characteristics, rDNA-ITS, and beta-tubulin-2 gene sequences. This is the first report of postharvest anthracnose on SMO caused by C. fioriniae.