Effect of controlled fluctuating low temperatures on survival of <Emphasis Type="Italic">Puccinia striiformis</Emphasis> f. sp. <Emphasis Type="Italic">tritici</Emphasis>

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is an important disease of wheat worldwide. Understanding the survival of Pst during the winter is critical for predicting Pst epidemics in the spring. We used a real-time quantitative PCR (qPCR) method to quantify Pst CYR32 biomass in infected wheat seedlings under several fluctuating temperature regimes (three average temperatures 0, ?5 and ?10 °C, each with two daily fluctuating amplitudes 8 and 13 °C). The survival of Pst CYR32 increased with increasing average temperature but also varied greatly with the amplitude – larger amplitude led to lower survival, particularly at 0 and ?5 °C. Nevertheless the survival at both amplitudes was still significantly greater than under the corresponding constant temperatures. There were small, albeit statistically significant, differences between the two cultivars (Xiaoyan 22, low winter-hardiness; Lantian 15, high winter-hardiness) in Pst CYR32 survival. This study indicated potential errors that could result from using daily average temperatures to predict Pst survival during the winter.     

An enrichment microsphere immunoassay for the detection of <Emphasis Type="Italic">Pectobacterium atrosepticum</Emphasis> and <Emphasis Type="Italic">Dickeya dianthicola</Emphasis> in potato tuber extracts

An enrichment microsphere immunoassay (MIA) was developed, based on the Luminex xMAP® technology, for the simultaneous (duplex) detection of Pectobacterium atrosepticum (former name Erwinia carotovora subsp. atroseptica) (Pca) and Dickeya dianthicola (former name Erwinia chrysanthemi) (Dcd) in potato plant extracts. Target bacteria in the extracts were enriched for 48 h in a semi-selective broth containing polypectate under low oxygen conditions. Samples were subsequently incubated with antibody-coated colour-coded microspheres (beads) and with secondary antibodies conjugated with Alexa Fluor® 532, a reporter dye. Samples were analyzed with the Luminex analyzer, in which one laser identified each microsphere and another laser the reporter dye conjugated to the secondary antibodies. The assay required minimal sample preparation, could be completed in 1 h, was performed in 96 wells microtitreplates and required no wash steps. The limit of detection for the duplex enrichment MIA was 100–1000 cfu ml^?1, which was a hundred times lower than of an enrichment-ELISA. Without enrichment, the sensitivity of MIA and ELISA was largely similar and ranged between 10⁶ and 10⁷ cells ml^?1. No difference in sensitivity was found between a MIA in a single or duplex format. In a comparative test with non-infected potato plant extracts and extracts from plants infected with Pca or Dcd, results of the enrichment MIA correlated well with those of the enrichment ELISA and enrichment PCR. These results indicate that MIA can be reliably used for multiplex detection of soft rot Enterbacteriaceae in crude potato plant extracts. The technology is an attractive and cost-effective alternative to other detection methods, including ELISA.     

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.     

<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.     

Visual detection of <Emphasis Type="Italic">Didymella bryoniae</Emphasis> in cucurbit seeds using a loop-mediated isothermal amplification assay

A method was developed using a Loop-mediated isothermal amplification assay (LAMP) for detecting Didymella bryoniae in cucurbit seeds. The LAMP primers were designed based on the DNA-dependent RNA polymerase II RPB140 gene (RPB2) from D. bryoniae. Calcein was used as an indicator for the endpoint visual detection of DNA amplification. The LAMP assay was conducted in isothermal (65 °C) conditions within 1 h. The detection threshold of the LAMP assay was 10 pg of genomic DNA and D. bryoniae was detected in 100 % of artificially infested seedlots with 0.05 % infestation or greater. With the LAMP assay, 16 of 60 watermelon and muskmelon seedlots collected from Xinjang province were determined to be positive for D. bryoniae. In contrast, a real-time PCR assay determined that 11 of the 60 seedlots from Xinjiang province were positive for the pathogen. These results showed that the LAMP technique was simple, rapid and well suited for detecting D. bryoniae DNA, especially in seed health testing.     

Intraspecific Evolution of <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> AG-1 IA Associated with Soybean and Rice in Brazil based on Polymorphisms at the ITS-5.8S rDNA Operon

Rhizoctonia solani AG-1 IA causes leaf blight on soybean and rice. Despite the fact that R. solani AG-1 IA is a major pathogen affecting soybean and rice in Brazil and elsewhere in the world, little information is available on its genetic diversity and evolution. This study was an attempt to reveal the origin, and the patterns of movement and amplification of epidemiologically significant genotypes of R. solani AG-1 IA from soybean and rice in Brazil. For inferring intraspecific evolution of R. solani AG-1 IA sampled from soybean and rice, networks of ITS-5.8S rDNA sequencing haplotypes were built using the statistical parsimony algorithm from Clement et al. (2000) Molecular Ecology 9: 1657–1660. Higher haplotype diversity (Nei M 1987, Molecular Evolutionary Genetics Columbia University Press, New york: 512p.) was observed for the Brazilian soybean sample of R. solani AG-1 IA (0.827) in comparison with the rest of the world sample (0.431). Within the south-central American clade (3-2), four haplotypes of R. solani AG-1 IA from Mato Grosso, one from Tocantins, one from Maranhão, and one from Cuba occupied the tips of the network, indicating recent origin. The putative ancestral haplotypes had probably originated either from Mato Grosso or Maranhão States. While 16 distinct haplotypes were found in a sample of 32 soybean isolates of the pathogen, the entire rice sample (n=20) was represented by a single haplotype (haplotype 5), with a worldwide distribution. The results from nested-cladistic analysis indicated restricted gene flow with isolation by distance (or restricted dispersal by distance in nonsexual species) for the south-central American clade (3-2), mainly composed by soybean haplotypes.     

Disease risk assessment of sugar beet root rot using quantitative real-time PCR analysis of <Emphasis Type="Italic">Aphanomyces cochlioides</Emphasis> in naturally infested soil samples

Sugar beet root rot, caused by the oomycete Aphanomyces cochlioides, is a serious and economically important disease of sugar beets world-wide. Today, disease risk assessment consists of a time-consuming greenhouse bioassay using bait plants. In the present study, a real-time quantitative PCR (qPCR) assay for determination of A. cochlioides DNA in field-infested soil samples was developed and validated using the standard bioassay. The qPCR assay proved to be species-specific and was optimized to give high amplification efficiency suitable for target copy quantification. A high correlation (R² > 0.98, p < 0.001) with pathogen inoculum density was shown, demonstrating the suitability for monitoring soil samples. The limit of detection (LOD) was evaluated in several different soil types and varied between 1 and 50 oospores/g soil, depending on clay content. Soils with a high LOD were characterised as having a low clay content and high content of sand. Varying levels of the A. cochlioides target sequence were detected in 20 of the 61 naturally infested soil samples. Discrepancies between the bioassay and the qPCR assay were found in soils from low- and medium-risk fields. However, the qPCR diagnostic assay provides a potentially valuable new tool in disease risk assessment, enabling sugar beet growers to identify high-risk fields.     

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.     

Egg and larval parasitoids of the beet armyworm<Emphasis Type="Italic">Spodoptera exigua</Emphasis> on maize in Turkey

The present study was conducted to determine egg and larval parasitoids of the beet armywormSpodoptera exigua Hübner (Lepidoptera: Noctuidae), which is an important but sporadic pest in Turkey. High beet armyworm population levels were recently observed in fields of first and second crop maize in the southeast Mediterranean region of Turkey. The parasitoid species complex and its impact on the pest were analyzed in a 4-year study in first and second crop maize. The braconid larval parasitoidsMicroplitis rufiventris Kokujev,M. tuberculifer Wesmael,Meteorus ictericus Nees,Chelonus obscuratus (Herrich Schäffer) (an egg-larval parasitoid),Apanteles ruficrus (Haliday); the ichneumonid larval parasitoidsHyposoter didymator (Thunberg) andSinophorus xanthostomus Gravenhorst; and the egg parasitoidTrichogramma evanescens (Westwood) were found to be the natural enemies attacking the pest. Among the parasitoid species the solitary endoparasitoidH. didymator was the most prevalent species, being reared from 40.5% of the parasitized larvae found. Higher parasitism rates were recorded on first crop than on second crop maize in every year. Possible reasons for this difference in larval parasitism between two growing seasons include lower population of the pest and reduced insecticide applications in first crop maize fields which permitted higher parasitism. However, parasitoid activity was insufficient to counterbalance the population growth of the pest on subsequent second crop maize.     

Description and molecular phylogeny of <Emphasis Type="Italic">Ditylenchus gilanicus</Emphasis> n. sp. (Nematoda: Anguinidae) from northern forests of Iran

Commercial areas containing Eucalyptus plantations have expanded in recent years due to increased demands for pulp, paper and bioenergy. One of the threats that can reduce Eucalyptus production is the eucalyptus rust disease caused by Austropuccinia psidii, a biotrophic fungus that affects a broad range of Myrtaceae. An accurate diagnosis tool for the early detection of rust disease could be useful in breeding programs for selection of resistant plants against rust, in phytosanitary purposes or in rust epidemics studies. The aim of the present work was to develop a SYBR Green-based quantitative real-time PCR (qPCR) assay for the early detection and quantification of A. psidii in Eucalyptus grandis leaves. Three sets of primers based on the A. psidii ribosomal DNA intergenic space region (IGS), beta-tubulin and elongation factor genes were designed and evaluated. The assays using the IGS primer set resulted in the highest detection efficiency, detecting a lower limit of 0.5 pg of A. psidii DNA. Under artificial inoculation in plants, A. psidii was detected immediately after pathogen inoculation until 240 h post-inoculation using qPCR. In field validation of the method, A. psidii was detected using qPCR in naturally infected leaves with or without rust symptoms. This easy and fast method can be used for an efficient detection of A. psidii in E. grandis leaves. The implications of this tool for rust studies are discussed below.     

Attempts to eradicate graft-transmissible infections through somatic embryogenesis in <Emphasis Type="Italic">Citrus</Emphasis> ssp. and analysis of genetic stability of regenerated plants

Ability to detect Pseudocercospora macadamiae infection in macadamia husk at least four months before symptoms become visible will aid the development of disease control measures. This study examined the distinctness of P. macadamiae within the phylogenetic lineages of the genus Pseudocercospora. In addition, we developed two quantitative PCR (qPCR) assays, as rapid diagnostic tools, for early detection and quantification of P. macadamiae in planta. Phylogenetic analysis of concatenated sequences of four gene loci (large subunits, internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF-1α) and actin of 47 P. macadamiae isolates showed that P. macadamiae is a distinct species in the genus Pseudocercospora. P. macadamiae isolates were partitioned into subunits in the cluster but the grouping of the isolates was regardless of location. Nucleotide diversity (0.02) and the coefficient of genetic differentiation (0.07) were low in the P. macadamiae population. Two qPCR primer sets, based on ITS (PMI) and TEF-1α (PME) were designed that consistently amplified P. macadamiae in fungal cultures (Ct = 16.93 ± 0.11 and Ct = 21.20 ± 0.11, respectively) and in planta (Ct = 32.36 ± 0.28 and Ct = 38.07 ± 1.20, respectively). The PMI primers also detected species in the genus Pseudocercospora, while PME was more specific and robust for quantification of P. macadamiae. Both primer sets detected P. macadamiae in asymptomatic tissue samples and strongly differentiated various stages of disease progression, which revealed approximately 10-fold increase in fungal biomass between each consecutive stage of symptom development.     

Evaluation and validation of a loop-mediated isothermal amplification test kit for detection of <Emphasis Type="Italic">Hymenoscyphus fraxineus</Emphasis>

Hymenoscyphus fraxineus (anamorph Chalara fraxinea) is the ascomycete fungus which causes ash dieback, a potentially lethal disease of Fraxinus excelsior in Europe. Isolation and culturing of H. fraxineus is time consuming and there is a need for rapid, specific diagnostic tools to assist in the deployment of appropriate phytosanitary measures. Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification method which can be used for on-site diagnosis. OptiGene have recently released a kit for rapid in planta detection of H. fraxineus using LAMP. The performance of the kit was evaluated for use in the laboratory and in the field in a comparison with real-time PCR, and the kit was also validated according to EPPO standard 7/98. The analytical sensitivity of the kit was found to be 7 pg of DNA extracted from pure culture. The kit was rapid, with an average time to positive of 15.5 min for field samples. A comparison of real-time PCR and LAMP was carried out in the laboratory and in the field. The diagnostic sensitivity and specificity of the LAMP kit in the laboratory were 90.2% and 98.0% respectively, and 98.3% and 88.6% in the field. Positive and negative predictive values and likelihood ratios were also calculated and considered in relation to the prevalence of the disease. The kit was found to be a useful diagnostic tool which can be applied in the field.     

Soil application of imidacloprid and related SAR-inducing compounds produces effective and persistent control of citrus canker

Soil application of the systemic insecticide imidacloprid (Admire®, Bayer Crop Science) produced season-long control of citrus canker caused by Xanthomonas citri sbsp. citri. Imidacloprid is a neo-nicotinoid that breaks down in planta into 6-chloronicotinic acid, a compound closely related to the systemic acquired resistance (SAR) inducer isonicotinic acid. Potted Swingle citrumelo seedlings (Citrus paradisi × Poncirus trifoliata) were treated with imidacloprid and the SAR inducers, isonicotinic acid, and acibenzolar-s-methyl as soil drenches or with acibenzolar-s-methyl as a foliar spray 1week prior to inoculation of immature leaves with X. citri sbsp. citri. Seedlings were re-inoculated four times over a 24-week period. SAR induction was confirmed by expression of the PR-2 gene (β-1,3 glucanase). Soil drenches of imidacloprid, isonicotinic acid, and acibenzolar-s-methyl induced a high and persistent up-regulation of PR-2 gene expression and reduced the number of canker lesions for up to 24 weeks compared to 4 weeks for foliar acibenzolar-s-methyl. Soil applied inducers of SAR reduced canker lesions up to 70% compared with the untreated inoculated plants. Lesions on leaves were small, necrotic, and flat compared to pustular lesions on inoculated untreated plants. Populations of X. citri sbsp. citri per leaf were reduced 1–3 log units in soil-treated plants compared to inoculated untreated plants.     

Inhibitory effect of <Emphasis Type="Italic">Lycium europaeum</Emphasis> extracts on phytopathogenic soil-borne fungi and the reduction of late wilt in maize

The ability to control soil-borne pathogens in agriculture is highly conditioned by the restricted use of synthetic pesticides. Allelopathy, the antimicrobial activity of plant extracts, is a promising option against crop pathogens. Extracts from Lycium spp. such as L. barbarum, L. chinense and L. intricatum possess biological and therapeutic properties. Individual methanolic extracts from leaves and stems of the Mediterranean medicinal species L. europaeum collected in two locations of Tunisia were each evaluated in vitro against Verticillium dahliae (Vd), Sclerotinia sclerotiorum (Ss) and Harpophora maydis (Hm). The mycelial growth of the three fungi was significantly reduced by all the extracts at doses of 10 and 30 μl mL^?1 (equivalent to 1 and 3 mg plant tissue mL^?1). The sporulation of Hm was almost completely inhibited in all the amendments, but that of Vd was stimulated by one of the leaf extracts when 1 and 3 mg dried plant tissue mL^?1 were used. Sclerotia of Ss were formed in a smaller number, their total weight increasing at extract doses equivalent to 1 mg plant tissue mL^?1 and higher. In greenhouse, the pathogenicity of Hm was confirmed as early as 6 weeks after inoculation, since it caused significant decreases of weights in both roots and aboveground parts of maize. The detrimental effect of Hm on maize root weight in greenhouse was significantly counteracted by one of the leaf extracts added by watering. In total, 11 phenolic compounds were separated in the four extracts. The hydroxycinnamic acid family, including chlorogenic acid as a major compound, represented more than 50% of the total content in all the samples. Rutin was the most abundant flavonoid. The results of this work show the detrimental effect of L. europaeum extracts against the soil-borne pathogens Hm, Ss and Vd, and highlight their potential in crop protection if adequately developed into final products and used in combination with other tools.     

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.     

<Emphasis Type="Italic">Dittrichia viscosa</Emphasis> and<Emphasis Type="Italic">Rubus ulmifolius</Emphasis> as reservoirs of aphid parasitoids (Hymenoptera: Braconidae: Aphidiinae) and the role of certain Coccinellid species

The role of the self-sown shrubsDittrichia viscosa (L.) W. Greuter andRubus ulmifolius Schott as reservoirs of aphid parasitoids was investigated. In the field studies conducted,D. viscosa grew adjacent to crops of durum wheat and barley andR. ulmifolius grew adjacent to cotton. The relative abundance of the parasitoids of(a) Capitophorus inulae (Passerini) onD. viscosa, (b) Rhopalosiphum padi (Linnaeus) on durum wheat and barley,(c) Aphis ruborum (Börner) onR. ulmifolius, and(d) Aphis gossypii Glover on cotton in various parts of Greece, was assessed during the years 1996–2000. In 2000, the fluctuation of parasitization of the above four aphid species was recorded and the action of the aphidophagous predators of the family Coccinellidae was studied. It was observed thatAphidius matricariae Haliday predominated onC. inulae andR. padi in all sampling cases. In contrast,Lysiphlebus fabarum (Marshall) was the dominant species parasitizingA. ruborum onR. ulmifolius andA. gossypii on cotton in Thessaly (central Greece) and Macedonia (northern Greece), whereasLysiphlebus confusus Tremblay et Eady andBinodoxys acalephae (Marshall) were the dominant parasitoid species in Thrace (northern Greece).Coccinella septempunctata Linnaeus was the most abundant coccinellid species on durum wheat, whereasAdonia variegata (Goeze) predominated on cotton. However, coccinellid individuals were scarce on bothD. viscosa andR. ulmifolius. The present study indicated that these two shrubs can be regarded as useful reservoirs of aphid parasitoids.     

Development of a TaqMan probe-based insulated isothermal PCR (TiiPCR) for the detection of <Emphasis Type="Italic">Acidovorax citrulli</Emphasis>, the bacterial pathogen of watermelon fruit blotch

Watermelon (Citrullus lanatus) is an important crop of the Cucurbitaceae family in fruit production worldwide. During its production, bacterial fruit blotch (BFB) caused by Acidovorax citrulli (Acidovorax avenae subsp. citrulli) is an important limiting factor on the volume and value of crops. This pathogen is known as a seed-borne pathogen, and the infested seeds can be a primary source of inoculum. Hence, a rapid and sensitive method for detecting A. citrulli on seeds would be an important tool in the management of BFB. In this study, we sought to develop a method to detect A. citrulli bacterial cells based on a TaqMan probe-based insulated isothermal PCR (TiiPCR) assay. Firstly, the specific primers and probe were designed based on a specific DNA fragment from the genome of A. citrulli. Then, PCR amplification was performed with the plasmid DNA to adjust the components of the PCR reagents, such as the concentrations of primers, magnesium chloride, and Taq DNA polymerase. Results revealed that 10 copies of plasmid DNA were detectable within the modified reagents by TiiPCR. Moreover, 10 bacterial cells in each reaction tube were detectable at a 100 % detection rate in this condition with a fluorescent signal intensification over 1.8. Based on these results, we concluded that a specific, rapid, and sensitive method based on TiiPCR had been successfully developed to detect bacterial cells of A. citrulli.