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.     

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.     

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.     

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.     

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.     

Adult attractiveness and oviposition preference of <Emphasis Type="Italic">Zabrotes subfasciatus</Emphasis> toward genotypes of common bean <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis>

The Mexican bean weevil Zabrotes subfasciatus (Coleoptera: Chrysomelidae: Bruchinae) is among the key pests of common bean Phaseolus vulgaris L. around the world. Identifying resistance sources and the resistance types involved is important for development of pest-resistant bean varieties. This study assessed the adult attractiveness and oviposition preference of Z. subfasciatus toward eight genotypes of common bean, including seven pre-selected resistant genotypes and one standard for susceptibility. Attraction to the genotypes was assessed at one and four days after infestation. Oviposition preference was tested under free- and no-choice (confinement) conditions. We observed that IAC 853 was the least oviposited genotype in the free-choice test. However, this finding was not confirmed by the no-choice test. Therefore, IAC 853 could not be characterized as resistant to Z. subfasciatus. Presence of the toxic protein arcelin does not appear to influence host selection by Z. subfasciatus.     

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.     

Molecular diversity in <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> isolates from common bean fields in Brazil

The common bean (Phaseolus vulgaris L.) is widely cultivated in Brazil and is known as a very important crop for families in this country. Fusarium wilt severely harms common beans and has become a big issue for this crop. In order to assist the breeding programs that target resistance to this disease, the evaluation of genetic diversity of the pathogen and its molecular characterization are crucial. Thus, the present goal was to identify Fusarium isolates obtained from several places in Brazil using molecular tools; select molecular markers for these isolates; and analyze their diversity. All of isolates were molecularly identified as Fusarium oxysporum f. sp. phaseoli (Fop). By using seven selected SSR markers, the results of diversity obtained by the dendrogram and the Bayesian analysis formed four groups where a large diversity of this fungus was found within each state. However, the groups were more homogenous according to the collection source and the pathogenicity test. More specifically, group 2 was composed of the most virulent strains and originated from Minas Gerais State – UFV, and group 3 was mostly composed by isolates from Goias state. Group I was also more diverse in terms of location and virulence. The overall results indicated a positive correlation between Fusarium diversity and its virulence to common bean. Furthermore, the use of these markers was effective in molecular identification and in detecting polymorphism within F. oxysporum f. sp. phaseoli.     

Fungicide resistance profile and genetic structure of <Emphasis Type="Italic">Botrytis cinerea</Emphasis> from greenhouse crops in Cyprus

Botrytis cinerea is a complex species prone to fungicide resistance and characterized by enormous genetic diversity. During 2013, 220 B. cinerea isolates causing gray mold were collected from greenhouse-grown crops in the regions of Ammochostos, Larnaca, and Limassol (Cyprus). Sensitivities of the sampled populations to seven botryticides with different modes of action were screened in vitro. The results of this in vitro screening highlighted the widespread phenomenon of fungicide resistance in greenhouses, since only 8.6 % of the isolates were sensitive to all botryticides. Resistance to thiophanate-methyl was the most prevalent, with frequencies ranging from 53.8 % to 80 %. Similarly, high resistance frequencies were observed for pyraclostrobin (27.1 to 78.9 %) and boscalid (28.2 to 66.2 %). Multiple fungicide resistant phenotypes were predominant, covering 67.3 % of the population, with frequencies of 80.0, 37.5, 53.8, 83.1, and 60.2 % in cucumber, eggplant, green bean, strawberry, and tomato, respectively. No fludioxonil-resistant isolates were observed. Botrytis cinerea and Botrytis group S genotypes comprised the gray mold population. B. cinerea was predominant within cucumber, eggplant and strawberry, whereas both genotypes were in equilibrium in green bean and tomato. However, Botrytis group S was found in all hosts. B. cinerea was the most prevalent in the majority of fungicide resistance phenotypes from strawberry, while genotype distributions within tomato were generally more balanced. B. pseudocinerea was not detected in the sampled population. Overall, frequency of the mating type allele MAT1–1 was higher to MAT1–2, underlying their unequal distribution in the population. However, cases of 1:1 distribution were apparent within particular subpopulations, suggesting that mating in the field cannot be excluded.     

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.     

Identification and partial characterization of a <Emphasis Type="Italic">Carica papaya</Emphasis>-infecting isolate of <Emphasis Type="Italic">Alfalfa mosaic virus</Emphasis> in Brazil

A Carica papaya plant with severe yellow leaf mosaic, leaf distortion, and systemic necrosis was found in the municipality of Piracicaba, state of São Paulo, Brazil. Transmission electron microscopy (TEM) analysis revealed the presence of potyvirus-like particles and bacilliform particles similar to those of the Alfamovirus genus. The potyvirus was identified as Papaya ringspot virus-type P (PRSV-P). Biological, serological, and molecular studies confirmed the bacilliform virus as an isolate of Alfalfa mosaic virus (AMV). Partial nucleotide and amino acid sequences of the coat protein gene of this AMV isolate shared 97–98% identity with the AMV isolates in the GenBank database. This report is the first of the natural infection of papaya plants by AMV.     

Five plant families support natural sporulation of <Emphasis Type="Italic">Cronartium ribicola</Emphasis> and <Emphasis Type="Italic">C. flaccidum</Emphasis> in Finland

Fusarium is one of the most destructive fungal genera whose members cause many diseases on plants, animals, and humans. Moreover, many Fusarium species secrete mycotoxins (e.g. trichothecenes and fumonisins) that are toxic to humans and animals. Fusarium isolates from date palm trees showing disease symptoms, e.g. chlorosis, necrosis and whitening, were collected from seven regions across Saudi Arabia. After single-sporing, the fungal strains were morphologically characterized. To confirm the identity of morphologically characterized Fusarium strains, three nuclear loci, two partial genes of translation elongation factor 1 α (tef1α) and β-tubulin (tub2), and the rDNA-ITS region, were amplified and sequenced. Of the 70 Fusarium strains, 70 % were identified as F. proliferatum that were recovered from six regions across Saudi Arabia. Fusarium solani (13 %), as well as one strain each of the following species: F. brachygibbosum, F. oxysporum, and F. verticillioides were also recovered. In addition, five Fusarium-like strains were recognized as Sarocladium kiliense by DNA-based data. The preliminary in vitro pathogenicity results showed that F. proliferatum had the highest colonization abilities on date palm leaflets, followed by F. solani. Although F. oxysporum f. sp. albedinis is the most serious date palm pathogen, F. proliferatum and F. solani are becoming serious pathogens and efforts should be made to restrict and control them. In addition, the potential toxin risks of strains belonging to F. proliferatum should be evaluated.     

Natural infection of <Emphasis Type="Italic">Nicandra physaloides</Emphasis> by <Emphasis Type="Italic">Tomato severe rugose virus</Emphasis> in Brazil

Nicandra physaloides, a common weed in South America, was found to be infected by an isolate of Tomato severe rugose virus (ToSRV), a bipartite begomovirus. The plants developed severe yellow rugose mosaic and were collected in São Paulo State, Brazil. This isolate of ToSRV was transmitted by Bemisia tabaci B biotype from infected plants of N. physaloides to healthy plants of N. physaloides and tomato in a glasshouse. This is the first report of natural infection of N. physaloides by ToSRV in Brazil.     

<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.     

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.     

Long-term dynamics of causative agents of stem base diseases in winter wheat and reaction of Czech <Emphasis Type="Italic">Oculimacula</Emphasis> spp. and <Emphasis Type="Italic">Microdochium</Emphasis> spp<Emphasis Type="Italic">.</Emphasis> populations to prochloraz

Trichoderma aggressivum is an aggressive contaminant mould in the cultivation of Agaricus bisporus leading to severe reductions in mushroom yields. Production of fully colonised A. bisporus substrate in Europe is commonly carried out in large tunnels (Phase III), after which the substrate undergoes several bulk handling (mixing) operations before ending up on shelves in mushroom growing facilities. The work presented here studied the effect of Trichoderma aggressivum inoculum, substrate mixing and supplementation on Agaricus bisporus yields and evaluated four methods to detect T. aggressivum in bulk handled substrate. Inoculum dilution level was shown to correlate well with mushroom yield (P < 0.0001) with reductions of 2–6 % at the most dilute level (10^?4) and 60–100 % at the most concentrated level (10^?1), depending on the experiment. Supplementation, with or without T. aggressivum, had no significant effect on mushroom yield (P ≥ 0.85) but a high degree of substrate mixing was shown to significantly increase (P < 0.0001) T. aggressivum-associated crop losses. Four T. aggressivum detection methods were evaluated and a quantitative polymerase chain reaction (qPCR) method gave the most consistent and least variable results. Cycle threshold (CT) values ranged from 24 to 40, depending on the experiment and the inoculum dilution level, and false negatives (CT = 40) were reported on one occasion with the most dilute samples. The results indicate that Phase III mushroom substrate is vulnerable to infection by T. aggressivum when the fully colonised substrate is broken up and mixed during bulk handling operations, identifying a previously unidentified risk for Phase III substrate producers.     

<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.