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.     

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.     

<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.     

Infection process of <Emphasis Type="Italic">Pseudocercospora musae</Emphasis> on banana leaf

Yellow Sigatoka that is caused by Pseudocercospora musae is an important banana disease. The aim of this study was to elucidate the infection process of P. musae in banana leaves by scanning electron microscopy. Leaf samples were inoculated on the abaxial surface with P. musae and then analysed at 12, 24, 36, 48, 72, 96, 120, 144, and 168 h post inoculation (hpi) and at 36 and 50 days post inoculation (dpi). The conidia were found to be germinated between 24 and 36 hpi and penetrated through the stomata between 96 and 120 hpi, or more generally from 144 hpi. P. musae colonized the spongy parenchyma at 36 dpi and the palisade parenchyma at 50 dpi. Sporulation occurred at 50 dpi on the adaxial surface of leaves through the emergence of conidia on conidiophores through the stomata. Considering the importance of yellow Sigatoka in banana production, our results provide a better understanding of the life cycle of the fungus for treatment processes.     

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.     

Rice response to simultaneous bacterial blight and drought stress during compatible and incompatible interactions

Plant response to one type of stress can be affected by simultaneous exposure to a second stress, for example when abiotic and biotic stresses occur together. Ten rice genotypes comprising those with bacterial blight (BB) resistance (R) genes, drought quantitative trait loci (QTLs) plus a BB R gene, and BB susceptible genotypes, were subjected to mild and moderate drought stress and plants were inoculated with two Xoo strains (PXO99 and PXO145) to simulate the challenges rice crops face under simultaneous stress of drought and BB. Plant height and dry shoot biomass were significantly reduced by drought stress treatments. The BB disease lesion lengths varied according to rice genotypes and PXO99 Xoo multiplication and spread in planta was higher compared to that of PXO145, which generally decreased under mild drought stress. Rice genotype IRBB7 (Xa7) showed less Xoo spread and a reduced Xoo multiplication under drought stress compared to the well-watered control with PXO145. In contrast, in genotypes with a different BB R gene and/or drought QTLs [IRBB4 (Xa4), IR87705–6-9-B (Xa4 + qDYT _2.2 ), IR87707–445-B-B-B (Xa4 + qDYT _2.2  + qDYT _4.1 ) and IR87707–446-B-B-B (Xa4 + qDYT _2.2  + qDYT _4.1 )], Xoo multiplication and spread in planta was higher with drought stress. This study has shown that drought stress affected rice response to the BB pathogen and the response varied according to the rice genotype. It is concluded that evaluating rice varieties under combined abiotic and biotic stresses will be the best strategy to determine biotic stress resistance durability under climate change.     

Effect of <Emphasis Type="Italic">Metarhizium guizhouense</Emphasis> infection on mating competition and mate choice of <Emphasis Type="Italic">Bactrocera latifrons</Emphasis> (Diptera: Tephritidae)

Metarhizium guizhouense PSUM02 treated males of Bactrocera latifrons were investigated for the mating competition among males and mating choice by female flies to develop an auto-dissemination for the control of B. latifrons. In the present study, on day 1–4 of experiment, M. guizhouense–treated male flies were equally competitive with the normal male flies as we did not observe any differences in mating by treated and normal male flies of B. latifrons. Further, mating competitiveness were found low in treated adult male B. latifrons than normal male B. latifrons from 5th days of treatment until death. Kaplan-Meier survival analysis of treated male flies gave average survival times (AST) of 4.3?±?0.1 days, while the healthy female and male flies in the same cage showed AST of 9.3?±?0.3 and 8.3?±?0.4 days, respectively. The AST of untreated flies in control experiment ranged from 14.2–14.5 days. In mating preference experiment, M. guizhouense–treated male flies were chosen by virgin female than gravid female flies for mating. The treated male flies caused mortality in both virgin and gravid female flies in the same cage with AST of 4.4?±?0.1, 5.6?±?0.1 and 7.4?±?0.2 days, respectively, while untreated flies showed AST ranged from 13.9–14.3 days in control. The treated male flies could transmit the fungus infection to both untreated female and male flies as well as in virgin and gravid female flies by mating and contact. Our experiments showed the potentiality of M. guizhouense PSUM02 in management of B. latifrons by auto-dissemination with treated male flies, which transmit the fungus to a healthy population to reduce insect pest infestations.     

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.     

<Emphasis Type="Italic">Aphelenchoides gorganensis</Emphasis> n. sp. (Nematoda: Aphelenchoididae)<Emphasis Type="Italic">,</Emphasis> a new species from Iran

Phytophthora capsici infection of chili pepper seedlings can cause substantial losses due to damping-off and collar rot diseases. Chemical control is no longer effective due to reported resistance development, on top of the related environmental concerns and the consumer demands for reduced use of fungicides. Biological control is a sustainable option, with several agents having been reported to be effective against this pathogen. This research focused on optimizing the application of strain THSW13 of Trichoderma hamatum and a bacterial isolate BJ10–86 with the objectives of improving chili pepper seed germination, reduce damping-off disease incidence, and improve the growth of the seedlings. Bacterial isolate BJ10–86 was subjected to molecular identification and found to be Pseudomonas aeruginosa. Chili pepper seeds treated with the biocontrol agents, individually or in combination, were seeded into commercial nursery media that had been pre-inoculated with P. capsici zoospores. Over a period of 35 days the chili pepper seed treatments significantly (P = 0.008) reduced the disease incidence of seedlings damping-off. Combined application of T. hamatum and P. aeruginosa was the best biocontrol treatment with an area under disease curve of only 36.61 units compared to 92.87 units for the control treatment. Similar results were observed in vitro where T. hamatum and P. aeruginosa synergistically inhibited P. capsici growth by 73.2 %. The inhibition activity of this treatment was similar to mefenoxam treatment, which implies that it is an effective and sustainable alternative for chili pepper seed treatment. The biocontrol seed treatment had no effect on seed germination and seedling growth.     

Morphological and biochemical basis of resistance in okra to cotton jassid, <Emphasis Type="Italic">Amrasca biguttula biguttula</Emphasis> (Ishida)

Fifteen okra germplasm entries viz. accessions: IC0506027, IC0506118 and EC0306728; Abelmoschus spp.: Abelmoschus tuberculatus, Abelmoschus moschatus, Abelmoschus angulosus, Abelmoschus tetraphyllus, Abelmoschus manihot and Abelmoschus caillei; genotypes: POL-6 and POL-7; and four cultivated varieties: Punjab 8, Punjab Padmini, Punjab 7 and Pusa Sawani were screened against jassid, Amrasca biguttula biguttula (Ishida) in field at Vegetable Research Farm, Department of Vegetable Science, Punjab Agricultural University, Ludhiana, India during Kharif 2015. Different morphological and biochemical parameters of leaves of the selected entries were also studied. The correlation between jassid nymphal population and mid vein hair density, total phenols and tannins was negative and significant (r = ?0.67, ?0.83, ?0.75, respectively); negative and non-significant for hair length, angle of insertion of hair, total sugars and silica (r = ?0.40, ?0.49, ?0.63 and ?0.59, respectively) and positive and highly significant for lacination index, reducing sugars and lignins (r = 0.82, 0.95 and 0.90, respectively). Abelmoschus spp. Abelmoschus tetraphyllus, Abelmoschus angulosus and Abelmoschus moschatus were found to be field resistant on the basis of significantly lower pooled jassid nymphal population (1.56–1.99), jassid injury index (1.16–1.27) and susceptibility index (2.70–2.92). High degree of resistance in Abelmoschus tetraphyllus, Abelmoschus angulosus and Abelmoschus moschatus was found to be associated with high hair density (4.75–7.50), longer hair (1285.00–1513.20 μm), more erect hair (83.40–95.20°), broad leaves, high total sugars (15.21–18.36 mg/g), total phenols (1.52–1.58 mg/g), tannins (26.12–31.48 mg/g) and silica (32.66–33.17 mg/g) and low levels of reducing sugars (2.50–3.39 mg/g). Abelmoschus tuberculatus, A. manihot, IC0506027 and EC0306728 were found moderately field resistant with variable levels of morphological and biochemical parameters. High hair density, broad leaves, moderate levels of total sugars, reducing sugars, total phenols, tannins and silica seems to be associated with moderate levels of resistance in these entries. The variable levels of above mentioned parameters in moderately resistant entries also indicate that a single factor is not responsible for resistance but combination of different factors may be conferring resistance to jassid.     

Challenging the larvae of <Emphasis Type="Italic">Helicoverpa armigera</Emphasis> and assessing the immune responses to nematode-bacterium complex

Helicoverpa armigera is a strong insecticidal resistance developed insect pest. The understanding of its innate immune responses to emerging biocontrol agent entomopathogenic nematode-bacterial complex can provide an opportunity to control this insect in an environmentally benign manner. Study was focused on role of hemocytes changes and PO activity in Steinernema abbasi-Xenorhabdus indica challenged larvae of H. armigera over the time. Total cell count changed effectively from 10.2?±?1.81?×?10⁵ to 15.5?±?3.3?×?10⁵ cells/mm³ upto 9 h and reduced distinctly up to 8.0?±?2.49?×?10⁵ cells/ mm³ in 24 h. PO activity inclined significantly and was recorded highest at 9 h (24.67?±?1.08?×?10² units) and lowest at 24 h (14.34?±?0.74?×?10² units) in total hemolymph with a similar pattern in plasma and the cellular fraction. Phenoloxidase activity in total and cellular component of hemolymph was positively correlated with prohemocytes, granulocytes and oenocytoids. Study showed the hemocytes and PO accounted as active immune responses against nematode infection. The results provide the first insight to understand the hemolytic activity, quick immunosuppression responses of S. abbasi-X. indica and vulnerability of H. armigera.     

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.     

Parasitism of <Emphasis Type="Italic">Psytallia concolor</Emphasis> (Hymenoptera: Braconidae) on <Emphasis Type="Italic">Bactrocera oleae</Emphasis> (Diptera: Tephritidae) infesting different olive varieties

Psytallia concolor (Szépligeti) is a koinobiont endoparasitoid of many Tephritidae larvae, including Bactrocera oleae (Rossi), and has been used in Mediterranean areas for biological control of olive fruit fly by inundative release. The present study evaluates the influence of olive fruit variety (Amfissis, Arbequina, Branquita de Elvas, Carolea, Kalamon, Koroneiki, Leccino, Manzanilla, Mastoidis, Moroccan Picholine and Picholine) on P. concolor parasitism efficiency and performance in the field during two successive years. The results showed that the percentage of parasitism was significantly higher (>30%) in Mastoidis and Koroneiki (light-weight varieties <1.5 g) than Leccino which has a medium fruit weight, followed by Amfissis, Moroccan Picholine, Picholine and Branquita de Elvas. Only Manzanilla among large weight varieties, exhibited high percentage of parasitization (42.72%) during 2013. The mean weight of the pupae (>4.21 mg) as well as the length of the developed adult parasitoids (>3.5 cm) in Mastoidis and Manzanilla were significantly higher than these individuals developed from other varieties such as Koroneiki and Kalamon. Finally, the optimal host fruit for P. concolor development seems to be Mastoidis variety with great biological parameters and percentage of parasitism.     

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.     

Relative potency of culture supernatants of <Emphasis Type="Italic">Xenorhabdus</Emphasis> and <Emphasis Type="Italic">Photorhabdus</Emphasis> spp. on growth of some fungal phytopathogens

In previous research, concentrated metabolites produced by bacteria of the genera Xenorhabdus and Photorhabdus (which are symbionts of entomopathogenic nematodes) were reported to be highly suppressive to fungal and oomycete plant pathogens. Conceivably, application of non-concentrated bacterial filtrates would be more economically feasible compared to using concentrated metabolites. We evaluated the potency of 10 % v/v cell-free supernatants of the bacteria X. bovienii, X. nematophila, X. cabanillasii, X. szentirmaii, P. temperata, P. luminescens (VS) and P. luminescens (K22) against Fusicladium carpophilum (peach scab), F. effusum (pecan scab), Monilinia fructicola (brown rot), Glomerella cingulata (anthracnose) and Armillaria tabescens (root rot). A bioactive compound derived from Photorhabdus bacteria, trans-cinnamic acid (TCA), was also compared with the bacterial filtrates. Fungal colony size based on manual measurements was compared for accuracy to measurements taken by image analysis. Supernatants of Xenorhabdus spp. exhibited stronger suppressive effects on spore germination and vegetative growth when compared with Photorhabdus spp. Overall, TCA was the most effective treatment; vegetative growth was completely inhibited by TCA (1.27 mg/ml). TCA treatments also suppressed spore germination of F. carpophylium and F. effussum by approximately 90 %. The efficacy of supernatants varied among Xenorhabdus species depending on the species tested, but X. szentirmaii filtrates tended to cause greater inhibition relative to the other bacteria supernatants. Manual measurement of colony diameter required at least two replicate estimates of the colony to avoid a type II error. Area measurements were slightly overestimated based on ruler measurements, but did not affect the outcome of the analysis. Supernatants of Xenorhabdus spp., Photorhabdus spp., or TCA, did not cause any phytotoxic effects when applied to various plant species in the greenhouse. Our results indicate the potential of using TCA or Xenorhabdus cell free supernatants as bio-fungicides. Such a product, based on bacterial culture supernatants, would be economically viable, marketable and easily applicable by the end-users in many situations.     

Vertical distribution of resting spores of <Emphasis Type="Italic">Plasmodiophora brassicae</Emphasis> in soil

Pratylenchus zeae parasitizes various crops and damages the host roots, resulting in decreased yield and quality of the host plants. Alignments of mitochondrial DNA (mtDNA) Cytochrome Oxidase I (COΙ) sequences revealed the genetic variation among Pratylenchus species. The results indicated 0.2–2.4% intraspecific variations for mtDNA COI sequences among eight P. zeae populations, and 25.4–35.1% interspecific variations between P. zeae and other Pratylenchus species. Based on the mtDNA COΙ region, a loop-mediated isothermal amplification (LAMP) assay was developed for the rapid and specific detection of P. zeae. The optimal conditions for the LAMP assay were 64 °C for 40 min. The LAMP products were confirmed using conventional polymerase chain reaction (PCR), analysis with the restriction enzyme Bam HI and visual inspection by adding SYBR Green I to the products. The LAMP assay could detect P. zeae populations from different hosts and different geographical origins specifically. The LAMP assay was also sensitive, detecting 0.1 individual P. zeae, which was 10 times more sensitive than conventional PCR. This is the first report of the detection of Pratylenchus spp. using LAMP. In addition, the results also suggested that use of the COI gene might allow for good resolution at the Pratylenchus species level.     

TaqMan PCR assay for detection and quantification of <Emphasis Type="Italic">Stagonosporopsis tanaceti</Emphasis> in pyrethrum seed and seedlings

Pyrethrum seed has an important role in the transmission of Stagonosporopsis tanaceti, the cause of ray blight disease of pyrethrum. A TaqMan probe based polymerase chain reaction (PCR) assay was developed to quantify the level of S. tanaceti inocula in pyrethrum seed and seedlings. Primer pair (St_qF3, St_qR2) was designed based on the intergenic spacer (IGS) region of S. tanaceti, which produced a 125 bp amplicon specific to S. tanaceti. TaqMan PCR assay using St_qF3, St_qR2 and a probe St_qP was highly specific against the genomic DNA of S. tanaceti, but did not amplify DNA of 14 related Stagonosporopsis species or other foliar pathogens of pyrethrum. The sensitivity limit of this assay was measured using the cycle threshold (C_t) value which ranged from 17.59 for 10 nanograms (ng) to 36.34 for 100 femtograms (fg) genomic DNA of S. tanaceti. There was a significant negative correlation (r = ?0.999, P < 0.001) between the C_t value and the percent of S. tanaceti infected seed. In addition, this TaqMan PCR assay detected latent infection within seedlings. This assay could be applied to test commercial seed and seedlings before deciding on the appropriate management practices.     

Estimation of Nuclear DNA Content and Determination of Relationship Between Altitude and Genome Size of USDA Turkish Oat (<Emphasis Type="Italic">Avena</Emphasis> spp.) Collection

Crop improvement is an important approach to overcome challenges raised from future uncertainties of agricultural systems and growing human population. The fundamental need for such improvement efforts is the availability of well characterized plant germplasm with sufficient genetic diversity. The 2C DNA is defined as the nuclear DNA content of an unreplicated diploid cell (in G1 phase) and is used both to get an estimate of genome size and ploidy level. Flow cytometry provides accurate and fast estimation of the genome size of plants. The genus Avena belongs to Poaceae (Gramineae) family and includes approximately 30 species including common oat (A. sativa). There are 837 Avena accessions in the USDA (United States Department of Agriculture) Germplasm Resources Information Network (USDA-GRIN) collected from Turkey. This also is the largest ex situ Avena collection from Turkey. However, initial genomic characterization of the collection has not yet been conducted. We estimated genome sizes and determined ploidy levels of Turkish oat collection. Nuclear DNA content of accessions ranged from 25.66 to 26.56?pg for A. sativa, from 25.48 to 26.88?pg for A. sterilis and from 24.85 to 26.41?pg for A. fatua. The average and range values for all three hexaploid species were in a similar close range. The range for putative tetraploid accessions belonging to A. barbata was from 12.79 to 16.90?pg. We found a number of aggregates. Furthermore, there was a negative correlation found between altitude and genome size. Obtained results will help to better utilize Avena collection in breeding efforts.     

A climatic risk analysis of the threat posed by the South American leaf blight (SALB) pathogen <Emphasis Type="Italic">Microcyclus ulei</Emphasis> to major rubber producing countries

In 2010, symptoms of cobweb disease were observed on cultivated Pleurotus eryngii crops in Spain. Based on morphological and genetic analyses, the causal agent of cobweb was identified as Cladobotryum mycophilum. Pathogenicity tests on fruit bodies were performed using conidial suspensions of three C. mycophilum isolates. The causal agent was re-isolated in 80–85 % of the fruit bodies inoculated internally and 15–40 % of those fruit bodies inoculated on the cap surface. The results pointed to a certain resistance of the P. eryngii cap surface to the mycelium of C. mycophilum. Two cropping trials inoculated with C. mycophilum were set up to evaluate the pathogenicity of the causal agent of cobweb in two casings. At the end of the growth cycle, 50–60 % of the inoculated blocks cased with mineral soil, and 20–33 % of the inoculated blocks cased with black peat showed cobweb symptoms. This difference in the appearance of the disease and its aggressiveness may be partly explained by different electrical conductivity values of the casing materials used. In vitro sensitivity of the C. mycophilum isolates and P. eryngii strains against four fungicides (chlorothalonil, prochloraz-Mn, thiabendazole and thiophanate-methyl) was assessed in radial growth experiments on fungicide-amended media. The most effective fungicides for inhibiting the in vitro growth of C. mycophilum were prochloraz-Mn and chlorothalonil, while prochloraz-Mn was also the most selective fungicide between P. eryngii and C. mycophilum, and chlorothalonil was the most toxic fungicide against the P. eryngii mycelium.     

Temperature and plant age drive downy mildew disease epidemics on oilseed <Emphasis Type="Italic">Brassica napus</Emphasis> and <Emphasis Type="Italic">B. juncea</Emphasis>

Studies were undertaken on the effects of temperature (14/10 °C and 22/17 °C day/night) and plant age (15, 23, 31 and 40 day-old-plants) on the severity of downy mildew (Hyaloperonospora parasitica) on oilseed Brassica cultivars (temperature: Brassica juncea Montara, B. napus Atomic, ATR-Hyden, Hyola 432, Hyola 450 TT, Thunder TT; plant age: B. juncea Dune, B. napus Surpass 402 and Hyola 450 TT). For temperature studies, there were significant (P?<?0.001) effects of temperature, cultivar, and cultivar x temperature interaction. On cotyledons of susceptible cultivars (B. napus Hyola 450 TT and Thunder TT), plants were symptomatic at 22/17 °C by 48 h post inoculation (hpi) and with abundant sporulation evident by 72 hpi, and with all cotyledons of B. napus Thunder TT collapsed by 7 days post inoculation (dpi). However, at 14/10 °C, there were no symptoms on the same cultivars until 5 dpi, and sporulation only observed at 7 dpi. Percent disease index values (DI%) at 22/17 °C of B. juncea Montara and B. napus ATR-Hyden, Hyola 432, Atomic, Hyola 450 TT and Thunder TT were 4.5, 49.0, 51.4, 65.8, 86.3 and 96.0, respectively, with all except B. juncea Montara having significantly lower (P?<?0.001) disease at 14/10 °C with DI% values of 2.8, 30.4, 27.9, 31.1, 44.4 and 76.4, respectively. For plant age studies, there were significant (P?<?0.001) effects of plant age, cultivar, and cultivar x plant age interaction. DI% was significantly higher at 15 compared to 40 day-old-plants (dop) across all cultivars. B. juncea Dune showed greatest resistance, particularly on 40 dop, with DI% values of 25.8, 24.6, 22.9 and 7.5, for 15, 23, 31 and 40 dop, respectively. B. napus Surpass 402 showed high susceptibility on cotyledons of 15 dop but moderate resistance on leaves of other ages, with DI% values of 59.0, 31.2, 27.1 and 26.2 for 15, 23, 31 and 40 dop, respectively. B. napus Hyola 450 TT showed very high susceptibility at the cotyledon stage on 15 dop, but some resistance on 23 dop and more so on 31 and 40 dop, with DI% values of 84.0, 41.2, 35.4 and 32.9 for 15, 23, 31 and 40 dop, respectively. Together, these findings explain for the first time why development of downy mildew epidemics on susceptible cultivars occurs early in the growing season when warmer seasonal temperatures in autumn coincide with presence of seedlings; in contrast to later in the growing season on less susceptible older plants coinciding with cooler and less favourable winter temperatures. Increasing maximum and minimum temperatures associated with climate change have likely fostered the increased severity of downy mildew over the past 15 years.