Molecular and experimental evidence of multi-resistance of <Emphasis Type="Italic">Cercospora beticola</Emphasis> field populations to MBC,DMI and QoI fungicides

Binucleate Rhizoctonia (BNR) spp. isolates were collected from taro (Colocasia esculenta (L.) Schott) and ginger (Zingiber officinale (Willd.) Roscoe) (Yunnanxiaojiang cv.) in Yunnan province. These Yunnan (YN) isolates did not anastomose with any of the tester isolates of the known AGs of binucleate Rhizoctonia spp. The growth of YN cultures on PDA was appressed, mealy and matlike after 4 days of incubation, then turned white brown, producing brown to dark brown, irregularly shaped sclerotia were embedded in the PDA medium after 14 days. All attempts to induce basidiospore production were unsuccessful, but the length and sequence of the internal transcribed spacer (ITS1 + 5.8S rDNA + ITS2) regions of 5.8S rDNA from the YN isolates were identical in length and sequence to isolates of all the other AGs of binucleate Rhizoctonia /Ceratobasidium spp. The sequences of 5.8S rDNA-ITS from the YN isolates were unique among AGs of BNR. The YN isolates had sequence similarities of 94% with isolates of AG Fb and P, 93% with AG E, 91% with AG R, 79–94% with AG S, and 74–87% with AG A, Ba, Bb, Bo, C, DI, DII, DIII, Fa, G, H, I, K, L, O, and Q. Four isolates of AG YN caused minor virulence (lesions ≦1mm²⁾ to ginger or taro in growth chamber studies. It was concluded that the YN isolates belong to a new anastomosis group AG-V of the Ceratobasidium spp..     

Pathological studies on the southern blight of China aster (<Emphasis Type="Italic">Callistephus chinensis</Emphasis>) caused by <Emphasis Type="Italic">Sclerotium rolfsii</Emphasis>

A severe outbreak of southern blight disease of China aster was observed during the post rainy season (September–November 2015) in the Mysore and Mandya Districts of Karnataka, Southern India. The disease incidence ranged between 12 and 47%. The typical disease symptoms include water-soaked lesions on leaves, stems and on the lower stem surfaces followed by quick wilting of the whole plant with abundant production of sclerotia near the stem-soil interface. The associated fungal pathogen was isolated on potato dextrose agar (PDA) medium, on which numerous reddish-brown sclerotia were seen. A total of 26 fungal isolates were isolated and studied for the mycelial compatibility. Isolate SrCCM 1 was used for pathogenicity analysis. The results of the study showed that, there was no variation among the isolates tested. Molecular identification of the pathogen by ITS-rDNA sequences of S. rolfsii showed 100% similarity with reference sequences. Based on the cultural, morphological and molecular characteristics, the fungal pathogen was identified as Sclerotium rolfsii Sacc. (Sexual morph: Athelia rolfsii (Curzi) C.C. Tu & Kimbr). Pathogenicity tests were performed on healthy leaves, roots and stems. Typical disease symptoms on leaves, stems and roots were evident after 5, 8 and 10 days of post-inoculation. Sclerotium rolfsii is known to cause diseases in economically important crop plants. However, no reports are available on the occurrence of S. rolfsii on China aster in India.     

Genetic structure of <Emphasis Type="Italic">Pyrenophora teres</Emphasis> f. <Emphasis Type="Italic">teres</Emphasis> and <Emphasis Type="Italic">P. teres</Emphasis> f. <Emphasis Type="Italic">maculata</Emphasis> populations from western Canada

Infection by Pyrenophora teres f. teres (Ptt) or P. teres f. maculata (Ptm), the causal agents of the net and spot forms of net blotch of barley, respectively, can result in significant yield losses. The genetic structure of a collection of 128 Ptt and 92 Ptm isolates from the western Canadian provinces of Alberta (55 Ptt, 27 Ptm), Saskatchewan (58 Ptt, 46 Ptm) and Manitoba (15 Ptt, 19 Ptm) were analyzed by simple sequence repeat (SSR) marker analysis. Thirteen SSR loci were examined and found to be polymorphic within both Ptt and Ptm populations. In total, 110 distinct alleles were identified, with 19 of these shared between Ptt and Ptm, 75 specific to Ptt, and 16 specific to Ptm. Genotypic diversity was relatively high, with a clonal fraction of approximately 10 % within Ptt and Ptm populations. Significant genetic differentiation (PhiPT = 0.230, P = 0.001) was found among all populations; 77 % of genetic variation occurred within populations and 23 % between populations. Lower, but still significant genetic differentiation (PhiPT = 0.038, P = 0.001) was detected in Ptt, with 96 % of genetic variation occurring within populations. No significant genetic differentiation (PhiPT = 0.010, P = 0.177) was observed among Ptm populations. Isolates clustered in two distinct groups conforming to Ptt or Ptm, with no intermediate cluster. The high number of haplotypes observed, combined with an equal mating type ratio for both forms of the fungus, suggests that P. teres goes through regular cycles of sexual recombination in western Canada.     

Characterization of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">melongenae</Emphasis> isolates from Turkey with ISSR markers and DNA sequence analyses

Fusarium oxysporum f. sp. melongenae (Fomg), causal agent of Fusarium wilt of eggplant, is a serious pathogen in open fields and greenhouses. Inter-simple sequence repeat (ISSR) banding profiles, sequence analyses of inter-transcribed-spacer (ITS), translation elongation factor 1-alpha (TEF-1α), and actin (actA) DNA regions were employed in this study to determine genetic diversity and population structure of Fomg isolates obtained from Turkey. For ISSR study, (ACTG)₅, (GACAC)₃, (GACA)₄, (GATA)₄, HVH(TG)₇ and (CA)₈RG primers were selected from a set of 16. Discriminative ability of the primers revealed with various indices including polymorphic information content (PIC), and mean PIC value was calculated as 0.26. The ISSR data revealed 31 loci belonging to 202 Fomg isolates and 14 of them were found to be polymorphic. The isolates on neighbor joining ISSR tree were grouped into two major clusters which separated Fomg and outgroup isolates. Population structure was investigated based on bayesian modeling and results indicated five subpopulations (K = 5, ?K = 205.42). Mean genetic and geographical distances among sampling locations revealed only a weak and insignificant correlation (r = 0.583, P = 0.06). Phylogenetic analyses were carried out with ITS, TEF-1α and actA DNA regions with a selected subset of 30 Fomg, along with one non-host and one outgroup isolates. Since ITS region were not able to provide a meaningful separation, TEF-1α and actA sequences of each organism were concatenated individually to build a dendrogram. The clustering tree successfully separated the Fomg, non-host and outgroup isolates in which all Fomg were located on the same branch, forming a monophyletic group in the dendrogram.     

Virulence profiling of <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis> isolates,causing bacterial blight of rice in India

Bacterial blight (BB) of rice caused by Xanthomonas oryzae pv. oryzae (Xoo), remains a major production constraint in rice cultivation especially in irrigated and rainfed lowland ecosystems in India. The pathogen is highly dynamic in nature and knowledge on pathotype composition among the Xoo population is imperative for designing a scientific resistance breeding program. In this study, four hundred isolates of Xoo collected from diverse rice growing regions of India were analyzed for their virulence and genetic composition. Virulence profiling was carried out on a set of differentials consisting of 22 near isogenic lines (NILs) of IR24 possessing different BB resistance genes and their combinations along with the checks. It was observed that different NILs possessing single BB resistance gene were susceptible to about 59–94% of the Xoo isolates except IRBB 13 (containing BB resistance gene xa13), which showed susceptibility to about 35% of the isolates. Based on the reaction of the Xoo isolates on the differentials, they were categorized into 22 pathotypes. Among the 22 pathotypes, IXoPt-1 and IXoPt-2 were least virulent and IXoPt # 18–22 were highly virulent. Pathotype IXoPt-19 which was virulent on all single BB resistance genes except xa13 constituted the major pathotype (22.5% isolates) and was widely distributed throughout India (16 states). This was followed by pathotype IXoPt-22 (17.25%) which was virulent on all the NILs possessing single BB resistance genes. Molecular analysis was carried out using two outwardly directed primers complementary to sequence of IS1112, a repetitive element of Xoo. A high level of genetic polymorphism was detected among these isolates and the isolates were grouped into 12 major clusters. The data indicated complex nature of evolution of the Xoo pathotypes and there was no strong correlation between pathotypes and genetic clusters as each genetic cluster was composed of Xoo isolates belonging to different pathotypes. The study indicated that none of the single BB resistance genes can provide broad spectrum resistance in India. However, two-gene combinations like xa5 + xa13 and different 3 or 4 genes combination like Xa4 + xa5 + xa13, Xa4 + xa13 + Xa21, xa5 + xa13 + Xa21 and Xa4 + xa5 + xa13 + Xa21 are broadly effective throughout India.     

Genetic variability within <Emphasis Type="Italic">Septoria carvi</Emphasis> Syd.- a pathogen of caraway <Emphasis Type="Italic">Carum carvi</Emphasis> L

Genetic variability within Septoria carvi isolates obtained from various organs of caraway cultivated in south-eastern and central Poland was studied using the RAPD-PCR technique. The tests were performed using randomly selected primers. The DNA profiles obtained using four primers proved useful in determining genetic variability among the genotypes of Septoria carvi isolates. The present study characterized the differences in the nucleotide sequence within the internal transcribed spacer region of rDNA (ITS1, 5.8S, ITS2) of selected S. carvi isolates and reference strains of Septoria spp. Moreover, eight isolates were sequenced for three loci: actin, calmodulin and translation elongation factor 1-alpha, and the obtained sequences were compared with the sequences of Septoria reference strains affecting other plants of the family Apiaceae. Phylogenetic analysis showed distinct differences of the tested isolates, which allowed to treat them Septoria carvi species affecting the above-ground organs of caraway Carum carvi L. This study is the first report on the genetic characteristics of the species S. carvi.     

Characterisation of <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> isolates from <Emphasis Type="Italic">Vitis vinifera</Emphasis> and potential biocontrol activity for the management of bitter rot of grapes

Aureobasidium isolated from Vitis vinifera (cv Chardonnay) grapevine tissues were characterised using morphological and molecular techniques. Species level identification of 29 isolates was accomplished by partial amplification and sequencing of the ITS region (ITS1–5.8S–ITS2) using universal primers ITS1 and ITS4. A comparison of nucleotide sequences using BLAST followed by phylogenetic analysis revealed that all isolates examined were Aureobasidium pullulans. Strain level discrimination of a total of 100 epiphytic Aureobasidium isolates including three reference strains was successfully carried out using two inter simple sequence repeat (ISSR) primers, (AAC)5 and (GTG)5 and the Intron Splice Junction R1 (ISJ-R1) primer in which 24, 24 and 15 scorable bands were produced for each primer, respectively. The high level of genetic variation recorded among the isolates further highlighted the high levels of strain diversity among A. pullulans residing on grapevines. Thirty-two epiphytic Aureobasidium isolates were examined for their ability to inhibit the growth of Greeneria uvicola, responsible for bitter rot of grapes. Using an in-vitro dual-culture antagonism assay, all isolates inhibited the growth of G. uvicola (Isolates DAR 77272 and DAR 77273) with inhibition ranging from 15 to 85%. Three Aureobasidium isolates were then examined for their ability to inhibit G. uvicola when co-inoculated onto detached berries, leaves and grape bunches growing on potted vines in a glass house. All isolates reduced the severity of bitter rot infection. The results indicate that A. pullulans has the potential to suppress bitter rot of grapes.     

Infection of <Emphasis Type="Italic">Sorghum bicolor</Emphasis>, selected grass species and <Emphasis Type="Italic">Zea mays</Emphasis> by G<Emphasis Type="Italic">loeocercospora sorghi</Emphasis>, causal pathogen of zonate leaf spot

Zonate leaf spot (Gloeocercospora sorghi) is a common disease in Sorghum bicolor producing areas of the U.S., but little is known about its biology, virulence and severity on S. bicolor, Zea mays, and related crop grassweeds. Greenhouse studies were conducted to determine and compare the virulence and severity of G. sorghi on 10 commercially available sorghum hybrids, four Z. mays hybrids and selected grassweed species including Sorghum bicolor (grain sorghum and shattercane biotypes) and Sorghum halepense (Johnsongrass), two of the most problematic arable weeds. Plants from the respective species were inoculated with a local G. sorghi isolate and maintained in a dew-chamber at 24 °C for 24 h and then incubated under greenhouse conditions for 4 weeks. Plants were observed for lesion expression and rated using a modified Horsfall-Barrett scale (0–10). The first symptoms of infection were visible within 24 h following inoculation on shattercane and S. bicolor hybrids. Symptoms consisted of small, non-diagnostic purple lesions on the leaves. Results showed that S. bicolor, S. halepense and shattercane were susceptible to G. sorghi. All other species tested in this study were not infected. More particularly, disease severity, increased from a rating of 3 to 10 on sorghum and from 2 to 7 on S. halepense between 2 and 23 days after inoculation, respectively. However, disease severity on shattercane increased rapidly from 3.5 to 10 between 2 and 8 days after inoculation, respectively. Among the sorghum hybrids tested, FFR-322 appeared to be the most resistant to G. sorghi while Pioneer 83G66 appeared to be the most susceptible. Z. mays hybrids were not infected by the fungus used in this study. G. sorghi could be used effectively to manage shattercane and S. halepense infestations occurring in Z. mays and S. bicolor fields consisting of specific G. sorghi-resistant hybrids.     

Characterization of <Emphasis Type="Italic">Myoviridae</Emphasis> and <Emphasis Type="Italic">Podoviridae</Emphasis> family bacteriophages of <Emphasis Type="Italic">Erwinia amylovora</Emphasis> from Hungary - potential of application in biological control of fire blight

The virulence spectrum of 300 isolates of Xanthomonas oryzae pv. oryzae (Xoo), representing 17 districts of Punjab, Pakistan was elucidated through inoculation on a set of six rice IRRI-differentials. The virulence level was assessed by using principal component and cluster analysis. Among six principal components (PCs), PC-1 exhibited 59.3 % of the total variance. The highly virulent isolates clusters on the positive side of the ordination away from the point of intersection of PC1 and PC2 and classifies the Xoo isolates from slow disease to the highest disease causing entities. The 300 isolates were categorized into 29 pathotypes (Pt1-29) wherein the highly virulent pathotype (Pt-1), comprises of 39 Xoo isolates were widespread in 12 districts. The majority of Xoo isolates were moderately to least virulent (21.7–43 %) and average disease progress curves confirmed the field reactions of these pathotype clusters for an efficient recognition of Xoo isolates. Interaction of the pathogen with differentials harboring different resistant genes was well investigated in the current study for future management approaches for which the surveillance of the new Xoo pathotypes may expedite the disease resistant rice breeding programme in the country.     

Influence of exogenous cholesterol in Pythiaceae resistance to inhibition by <Emphasis Type="Italic">Trichoderma</Emphasis> antibiosis

The purpose of this study was to determine if exogenous cholesterol availability influenced Pythiaceae resistance to antibiosis. Characterisation of an isolate of Phytophthora erythroseptica and Pythium ultimum for tolerance to antibacterial compounds found that 0.05 g.l^?1 chloramphenicol inhibited mycelial growth by 96.6 % and 23.5 % respectively. However, the addition of cholesterol (0.01 g l^?1) to potato dextrose agar (PDA) containing 0.05 g l^?1 chloramphenicol was found to increase mycelial growth of P. erythroseptica, indicating a role for cholesterol in tolerance to inhibitory antibacterial compounds. To determine if this property extended to suppressive effects of a potential biocontrol agent, P. erythroseptica and P. ultimum were then tested against a cell-free filtrate of diffusible metabolites produced by a suppressive Trichoderma harzianum isolate in the presence and absence of cholesterol in PDA. In the absence of cholesterol, diffusible metabolites of the T. harzianum isolate were found to inhibit mycelial growth of P. erythroseptica and P. ultimum on PDA by 98 % and 63.6 % respectively (P?<?0.0001). However, the inhibitory effect of the metabolites was mitigated when 0.005 g l^?1 of cholesterol was present in PDA, with mycelial growth of P. ultimum and P. erythroseptica reduced by only 60.4 % and 41.8 %, respectively (P?<?0.0001), much less inhibition than was observed in the absence of cholesterol. These results demonstrated that access to exogenous cholesterol can influence the sensitivity of Pythiaceae species to antibiosis by positively influencing mycelial growth.     

Population features of <Emphasis Type="Italic">Xanthomonas arboricola</Emphasis> pv. <Emphasis Type="Italic">pruni</Emphasis> from <Emphasis Type="Italic">Prunus spp.</Emphasis> orchards in northern Italy

Bacterial leaf/fruit spot and canker of stone fruits, caused by Xanthomonas arboricola pv. pruni, is a recurrent disease in Italy. A set of 23 strains has been isolated in peach and plum orchards in an intensively stone fruit cultivated area located in north-eastern Italy. They were all identified as X. arboricola pv. pruni by means of phytopathological and serological features: hypersensitive reaction on bean pods, pathogenicity test on immature peach or plum fruitlets, identification by immunofluorescence assay and conventional PCR. Phylogenetic analysis based on sequencing of the gyrB housekeeping gene of the isolates showed that they formed a unique clade, well characterised and separated from other xanthomonads. An insight into the genetic population features was attempted by rep-PCR analysis, using the ERIC, REP and BOX primers. The combined rep-PCR fingerprints showed a slight intra-pathovar variation within our isolates, which grouped in five close clusters. Copper resistance has been assessed in vitro for our whole X. arboricola pv. pruni collection, highlighting that two isolates show a level of resistance in vitro up to 200 ppm of copper. Nonetheless, the copLAB gene cluster, present in many other species of Xanthomonads, was not detected in any isolate, confirming the presence of a still unknown mechanism of copper detoxification in our Xanthomonads arboricola pv. pruni tolerant/resistant strains.     

<Emphasis Type="Italic">Bradyrhizobium</Emphasis> isolated from huanglongbing (HLB) affected citrus trees reacts positively with primers for <Emphasis Type="Italic">Candidatus</Emphasis> Liberibacter asiaticus

Bradyrhizobium sp., a slow-growing nitrogen-fixing symbiotic bacterium of legumes and common root endophyte of other plants, is closely related to Candidatus Liberibacter asiaticus (Las), the uncultured putative pathogen associated with citrus huanglongbing (HLB). In attempts to isolate Las on a low-nutrient medium that had been used for the isolation of several uncultured bacteria of the alpha subclass of proteobacteria, slow-growing Bradyrhizobium spp. were isolated and identified by sequencing of 16S rDNA. The individual isolates tested weakly positive (Ct = 31.2–36.0) with the USDA primers commonly used in qPCR assays for Las in foliar tissues. Direct DNA extracts from roots of HLB symptomatic trees that contained sequences of Bradyrhizobium sp. had Ct values ranging from 31.2 to 36.5; sequences of Las were not present in those samples. Potential cross-reaction between DNA of members of the Rhizobiales and sequences amplified by the Las primers were tested in silico with the Primer-BLAST tool in NCBI. Similar to Las, Bradyrhizobium generated predicted 16S rDNA amplicon sizes of 78–79 bp with the qPCR primers and of 1167-1172 bp with the conventional PCR primers. Bradyrhizobium sequences of 16S rDNA had 1–7 mismatches and only 1 mismatch at the 3′ end of qPCR and conventional PCR primers confirming potential cross-reactivity. As Bradyrhizobium is usually not found in foliage, the USDA qPCR primers can be safely used to check leaves for the presence of Las, but a threshold value of 31.0 is recommended for Las detection in roots. Other primers should be tested for potential cross-reaction with members of the Rhizobiales.     

Identification and characterization of <Emphasis Type="Italic">Alternaria alternata</Emphasis> (Fr.) Keissler causing <Emphasis Type="Italic">Ceratonia</Emphasis> Blight disease of carob (<Emphasis Type="Italic">Ceratonia siliqua</Emphasis> L.) in Turkey

A new dagger nematode, Xiphinema tica n. sp., is described and illustrated from several populations extracted from soil associated with several crops and wild plants in Costa Rica. The new dagger nematode is characterised by a moderate body size (3276–4240 μm), a rounded lip region, ca 13.5 μm wide, separated from body contour by a shallow depression, amphidial fovea large, stirrup-shaped, a moderately long odontostyle ca 135 μm long, stylet guiding ring located at ca 122 μm from anterior end, vulva almost equatorial (50–54%), well-developed Z-organ, with heavy muscularised wall containing in the most of specimens observed two moderately refractive inclusions variable in shape (from round to star-shaped), with uterine spines and crystalloid bodies; female tail short, dorsally convex-conoid, with rounded end and a small peg, with a c’ ratio ca 0.8, bearing two or three pairs of caudal pores and male absent. The unique and novel uterine differentiation based on the coexistence of a well-developed Z-organ mixed with uterine spines and crystalloid bodies in Xiphinema prompted us to update and include this combination of characters in the polytomous key of Loof and Luc (1990). Integrative diagnosis was completed with molecular data obtained, using D2-D3 expansion segments of 28S rDNA, ITS1-rDNA, partial 18S–rDNA and the partial mitochondrial gene cytochrome c oxidase subunit 1 (coxI). The phylogenetic relationships of this species with other Xiphinema spp. indicated that X. tica n. sp. was monophyletic to the other species from the morphospecies Group 4, Xiphinema oleae.     

<Emphasis Type="Italic">Fusarium ershadii</Emphasis> sp. nov., a Pathogen on <Emphasis Type="Italic">Asparagus officinalis</Emphasis> and <Emphasis Type="Italic">Musa acuminata</Emphasis>

Two Fusarium strains, isolated from Asparagus in Italy and Musa in Vietnam respectively, proved to be members of an undescribed clade within the Fusarium solani species complex based on phylogenetic species recognition on ITS, partial RPB2 and EF-1α gene fragments. Macro- and micro-morphological investigations followed with physiological studies done on this new species: Fusarium ershadii sp. nov can be distinguished by its conidial morphology. Both isolates of Fusarium ershadii were shown to be pathogenic to the monocot Asparagus officinalis when inoculated on roots and induced hollow root symptoms within two weeks in Asparagus officinalis seedlings. In comparison mild disease symptoms were observed by the same strains on Musa acuminata seedlings.     

Comparative colonisation by virulent versus avirulent <Emphasis Type="Italic">Pyricularia oryzae</Emphasis> on wild <Emphasis Type="Italic">Oryza australiensis</Emphasis>

Pyricularia oryzae (rice blast) conidial development at pre-penetration stage determines success or otherwise of infection inside the rice host plants. Studies on conidial germination and growth on the leaf surface in commercial rice (Oryza sativa) report differently, dependent upon host type and level of blast resistance. Although wild rice (O. australiensis) is known to be an alternative host of blast, the interaction between P. oryzae conidia and wild O. australiensis on its leaf surface has not been previously studied. We found significant (P?<?0.001) differences in conidial development between two blast isolates with different virulence in terms of conidial germination, germ tube growth and appressoria formation on both wild and cultivated rice. Conidial germination at 6 h post-inoculation (hpi) for the virulent isolate was significantly (P?<?0.001) delayed. Germ tubes of the avirulent isolate conidia grew significantly (P?<?0.001) faster and with significantly (P?<?0.001) longer germ tubes than from virulent conidia. Appressoria development for the virulent isolate was significantly (P?<?0.001) faster at its later growth stages of 12 and 18 hpi when approximately 100% of germ tubes formed appressoria. In contrast, formation rate of appressoria for the avirulent isolate was significantly (P?<?0.001) slower and only reached 76% of germ tubes forming appressoria. Appressoria formation on O. australiensis was significantly (P?<?0.001) greater than the formation on O. sativa for both virulent and avirulent P. oryzae at 12 hpi, a clear indication that host type influences the extent of appressoria formation.     

Competence of <Emphasis Type="Italic">Frankliniella occidentalis</Emphasis> and <Emphasis Type="Italic">Frankliniella intonsa</Emphasis> strains as vectors for <Emphasis Type="Italic">Chrysanthemum stem necrosis virus</Emphasis>

The vector competence of Frankliniella occidentalis for Chrysanthemum stem necrosis virus (CSNV) was evaluated. Three vector strains with distinct competences for Tomato spotted wilt virus (TSWV) transmission were investigated, including an artificially selected strain (TsH) that has a particularly high competence (>90 %). Newly hatched larvae of F. occidentalis were given an acquisition access period of 5 days on CSNV-infected D. stramonium leaves, and reared to maturity. Their transmission efficiencies were examined using a leaf disk assay using Petunia x hybrida leaves. Following the leaf disk assay, the virus accumulation in the vectors was examined via a double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) of their bodies. The results showed that the CSNV acquisition and transmission efficiency of the TsH strain did not differ from those of the others, indicating that the competence of F. occidentalis as a vector for CSNV is not related to that for TSWV. The CSNV transmission and acquisition efficiencies of two F. intonsa strains (Hiroshima and Fukuoka) were also evaluated. In Hiroshima strain, 35 % of adults were viruliferous, but only two transmitters (3 %) were observed. In Fukuoka strain, 6 % were viruliferous, and no transmitters were observed. These results indicate that F. intonsa cannot be a major vector for CSNV. The accumulation of CSNV in the adults of F. occidentalis and F. intonsa evaluated using DAS-ELISA showed a significant difference in ELISA values among transmitter, viruliferous non-transmitter, and non-viruliferous individuals. These results clearly demonstrated that only transmitters that accumulated a threshold quantity of virus can transmit CSNV to plants.     

Bacterial canker of cherry trees, <Emphasis Type="Italic">Prunus avium,</Emphasis> in South Africa

Laboratory and nursery experiments were conducted to identify the causal agent of a needle blight of Pinus wallichiana, a species native to the Western Himalayas. The pathogen was identified as Myrothecium verrucaria, on the basis of morphological, cultural and molecular characterization. BLAST analysis of ITS sequences of the pathogen revealed maximum sequence identity of 99% with M. verrucaria. The sequence is the first of this fungus from P. wallichiana. Phylogenetic analysis grouped all M. verrucaria isolates in a single clade; M. roridum and M. inundatum clustered in separate clades. The pathogen grew optimally at 25 ± 1 °C on oat meal agar, pH 5.5. Inoculation experiments with M. verrucaria demonstrated pathogenicity on Pinus halepensis, Cedrus deodara and Cryptomeria japonica, in addition to Pinus wallichiana.     

Morphological and molecular characterization of <Emphasis Type="Italic">Diaporthe (</Emphasis>anamorph <Emphasis Type="Italic">Phomopsis)</Emphasis> complex and pathogenicity of <Emphasis Type="Italic">Diaporthe aspalathi</Emphasis> isolates causing stem canker in soybean

The complex of Diaporthe (asexual morph) species occurring on soybean constitutes an important pathogenic group associated with diseases such as pod and stem blight, seed decay and stem canker. Stem canker, caused by Diaporthe aspalathi, has been reported as the most aggressive form of canker and its occurrence has limited soybean crop productivity in the southern United States. The main form of pathogen control is the use of stem canker resistant soybean varieties. In this study, strains of Diaporthe and Phomopsis were isolated from stem and seeds of soybean in different locations in South America during the years 1989–2014. Genomic DNA from 26 isolates were analyzed by PCR-restriction fragment length polymorphism (RFLP) and Amplified Fragment Length Polymorphism (AFLP) techniques, and sequencing of internal transcribed spacer (ITS) regions of ribosomal DNA. The molecular analysis of ITS sequences by alignment with those of ex-type strains deposited in GenBank and morphological characteristics allowed the identification of Phomopsis longicolla, D. phaseolorum var. sojae, D. caulivora and D. aspalathi. An analysis of the pathogenicity of 13 isolates of D. aspalathi inoculated in soybean genotypes carrying different resistance genes to stem canker (Rdm1, Rdm2, Rdm3, Rdm4, Rdm5 and Rdm?) enabled us to identify the occurrence of at least three races of D. aspalathi occurring in Brazil. Among the isolates identified as D. aspalathi, both molecular and phenotypic analyses showed clustering depending on the date of collection and pathogenicity, which revealed the existence of variability of the pathogen.     

Characterization of <Emphasis Type="Italic">Bean common mosaic virus</Emphasis> and <Emphasis Type="Italic">Bean common mosaic necrosis virus</Emphasis> isolates in common bean growing areas in Turkey

Bean common mosaic virus (BCMV) and Bean common mosaic necrosis virus (BCMNV) are well-known legume-infecting potyviruses. The incidences of BCMV and BCMNV infections were determined by ELISA in 367 seed and leaf samples which were collected in 15 common bean-growing provinces of Turkey. Of the samples tested, 67 (18.2 %) occurred to be infected with BCMV, however only 5 (1.4 %) were infected with BCMNV. A total of 45 ELISA-positive samples were selected from single-virus infected ones to determine BCMV and BCMNV pathogenicity groups (PGs) by using a set of bean cultivars that contain different combinations of resistance genes. Some BCMV populations exhibiting unusual pathogenicity were identified. One of them, named TR-180, was found to overcome resistance conferred by bc-1, bc-1 ² , bc-2 and bc-2 ² recessive alleles in common bean and assigned to PG VII. This isolate shared high (99 %) sequence identity with previously identified BCMV RU-1 and RU-1-related strains (RU1-OR-B and RU1-OR-C) according to a BLAST analysis of the nucleotide sequences of RT-PCR amplified products comprising the complete coat protein and 3′ partial NIb regions. The isolates TR-203 and TR-256 produced a distinctive reaction pattern in the dominant I gene-bearing bean cultivars Amanda and Isabella at lower (<30 °C) temperatures and were classified into PG IVb. These isolates were found to be 99 % identical to US-1 strain based on 3′ terminal nucleotide sequences of the BCMV genome. A fourth isolate, TR-243, involved mixed BCMV populations, as confirmed by partial nucleotide sequence analysis; one was classified as belonging to PG VII being similar to TR-180, and another was assigned to PG IVb. In conclusion, on the basis of both the reactions of differential bean cultivars and ELISA results, most of BCMV isolates were assigned to pathogroup PG VII and BCMNV isolates to PG VIb. This study is the first to show that four recessive resistance alleles of common bean can be overcome by a single field isolate of BCMV, and that a wide range of BCMV pathogroups are present in Turkey.     

Antifungal activity of <Emphasis Type="Italic">Boerhavia diffusa</Emphasis> L. extract against <Emphasis Type="Italic">Phytophthora</Emphasis> spp. in tomato and pepper

A new longidorid nematode, Longidorus persicus n. sp., is described and illustrated from a population extracted from soil associated with wild rose (Rosa sp.) naturally growing in the mountains close to the village of Cheshmeh-e-Nezamei near the city of Gilan-e-Gharb, Kermanshah province, western Iran. The new needle nematode is characterised by a large body size (6550–7763 μm), an anteriorly flattened lip region, ca 13 μm wide, distinctly set off from body contour by a depression, amphidial fovea large, pocket shaped, slightly asymmetrically bilobed, a moderately long and flexible odontostyle ca 86 μm long, stylet guiding ring located at ca 25 μm from anterior end, posterior arrangement of the pharyngeal gland nuclei, vulva almost equatorial (49–54 %), tail short, about 3/4 of its width, dorsally convex-conoid, with rounded terminus, with a c’ ratio ca 1.3, bearing three pairs of caudal pores and male rare (ratio 1:10 females) with spicules ca 46 μm long. Integrative diagnosis was completed with molecular data obtained using D2-D3 expansion segments of 28S rDNA, ITS1-rDNA, and partial 18S-rDNA. The phylogenetic relationships of this species with other Longidorus spp. using D2-D3 expansion segments and partial 18S-rDNA indicated that L. persicus n. sp. clustered together with L. perangustus and L. euonymus: both of them sharing an anteriorly flattened lip region and distinctly set off from body contour by a depression.