Accumulation of toxigenic <Emphasis Type="Italic">Fusarium</Emphasis> species and <Emphasis Type="Italic">Stenocarpella maydis</Emphasis> in maize grain grown under different cropping systems

Entomopathogenic nematodes in the genus Steinernema are associated with Xenorhabdus spp. bacteria. When steinernematid colonise an insect host the nematode-bacterium association overcomes the insect immune system and kills the host within 48 h. Xenorhabdus spp. produce secondary metabolites that are antifungal to protect nematode-infected cadavers from fungal colonization. The concentrated, or cell-free metabolites of X. szentirmaii exhibit high toxicity against various fungal plant pathogens and show potential as natural bio-fungicides. In the current study, we determined 1) thermo-stability, 2) dose-response, and 3) shelf-life of antifungal metabolites of X. szentirmaii against Monilinia fructicola (cause of brown rot of peach and other stone fruit) and Glomerella cingulata (cause of antharacnose). Thermo-stability was determined by autoclaving bacterial culture broths (121 °C and 15 psi for 15 min) and measuring fungal growth on in potato dextrose agar (PDA) containing 10% of the supernatants. Autoclaving had no impact on the antifungal activity of the secondary metabolites. Over a test period of 9 months, the activity of both extract types did not decline when stored at 4 or 20 °C. A dose-response study (10, 20, 40, 60, 80 and 100% supernatant-containing metabolite) using both phytopathogens demonstrated that a greater dose of supernatant increased antifungal activity. The antifungal-metabolite containing supernatant of X. szentirmaii has potential as a bio-fungicide. These results demonstrate the metabolite(s) are thermo-stable, they have a long shelf-life and require no stabilizing formulation, even at room temperature.     

Antifungal activity of the essential oil of Zanthoxylum bungeanum and its major constituent on Fusarium sulphureum and dry rot of potato tubers

The composition of the essential oil of Zanthoxylum bungeanum was analyzed by gas chromatography-mass spectrometry (GC-MS). A total of 35 components, which constitute 99.10% of the oil, were identified. The major constituent was α-pinene (19.59%). Minimum inhibitory concentrations (MICs) of the essential oil and α-pinene against Fusarium sulphureum were determined to be 6.25% and 12.50%, respectively. A determination of the antifungal activity and inhibitory effect of the essential oil and α-pinene showed that the essential oil more significantly decreased cell membrane integrity and inhibited spore germination than α-pinene. Observations using scaning electron microscope revealed morphological modification including mycelium asymmetry, hyphal swelling, curling, and a secretory appearance. Ultrastructural alterations were also observed including cell empty cavity and osmiophilic granule generation. In vivo testing showed that the oil and α-pinene effectively controlled dry rot of potato inoculated with F. sulphureum. These findings indicated that the oil and α-pinene directly inhibit fungal growth, and the essential oil showed pronounced potential than α-pinene.     

Phytosynthesis of Silver Nanoparticles using Leaf Extracts from <Emphasis Type="Italic">Ocimum basilicum</Emphasis> and <Emphasis Type="Italic">Mangifira indica </Emphasis>and their Effect on some Biochemical Attributes of <Emphasis Type="Italic">Triticum aestivum</Emphasis>

In this investigation, leaf extracts of Ocimum basilicum and Mangifira indica were used as reducing agents for biosynthesis of silver nanoparticles (AgNPs). The biosynthesized AgNPs were authorized by UV-vis spectrophotometry and X?ray diffraction (XRD) analysis. AgNPs were obtained after 5?min of reaction at 80^oC. The formation of AgNPs was confirmed by the presence of absorption peaks at 439?nm using extract from O. basilicum and at 442.5?nm from M. indica. X?ray spectra showed strong peaks for the crystalline Ag. Shape and size of the biosynthesized AgNPs were studied using high resolution transmission electron microscope (HR-TEM). Size of the produced AgNPs was found to be 9–35?nm. Effect of the synthesized nanosilver was then investigated on some biochemical attributes of wheat plant (Triticum aestivum cultivar saka 92). The growth parameters such as shoot lengths, fresh and dry weight of shoot, chlorophyll, carbohydrate and protein contents in shoot of wheat plant were investigated. Application of AgNPs synthesized from Ocimum basilicum and from Mangifira indica at the concentrations of 20,40?ppm showed an increase in shoot length, fresh and dry weight of shoot, chlorophyll, total carbohydrate and protein content in shoot of wheat plants, beyond these concentrations an inhibitory effects were shown.     

Pre-harvest root application of soluble silicon improves postharvest quality and induces antifungal compounds in bitter gourd (<Emphasis Type="Italic">Momordica charantia</Emphasis> L.)

Beneficial effects of Silicon (Si) on disease control have been shown in a number of plant pathosystems. The present study ascertained the effect of root application of soluble Si on postharvest quality of bitter gourd (Momordica charantia L.) with special reference to the disease development by Fusarium oxysporum. Plants were treated with Potassium silicate (Si+) by amending the soil mix (200 mgSi /kg soil). Treatments were started at the four leaf stage and continued up to 28 days at seven days intervals. Soil amendments with Si significantly reduced the postharvest weight loss and extended the shelf life by 2 days through delayed ripening and reduced fungal rot development. Si analysis in bitter gourds, harvested from Si?+?plants revealed that fruit tissues accumulated nearly1.5 times as much silicon (0.54% dry weight) as those grown in control (Si-) mix (0.36% dry weight). Extracts from Si?+?plants exhibited a stronger antifungal activity on thin layer chromatography (TLC) against Cladosporium cladosporioides. Methanolic extracts of fruit pericarp separated on TLC plates showed four antifungal zones at Rf 0.17, 0.51, 0.64 and 0.93. Compound separated at Rf 0.64 was observed in samples from Si?+?and/or challenged with F.oxysporum. Antifungal zone at Rf 0.51 was present only when Si?+?fruits were challenged with the fungus. These results suggest that by using soil application of soluble Si, postharvest quality of bitter gourd can be improved and shelf life can be enhanced by 2 days compared to the control. Enhanced fungitoxic activity may have some role in Si-induced disease resistance againstpostharvest fungal pathogens.     

Nonhost resistance of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> against <Emphasis Type="Italic">Colletotrichum</Emphasis> species

Arabidopsis thaliana exhibits a durable resistance called nonhost resistance against nonadapted fungal pathogens. A. thaliana activates preinvasive resistance and terminates entry attempts by nonadapted fungi belonging to the genus Colletotrichum, which cause anthracnose disease in many plants. In the interaction between A. thaliana and nonadapted C. tropicale, the preinvasive resistance involves the PENETRATION 2-related antifungal secondary metabolite pathway and the ENHANCED DISEASE RESISTANCE 1-dependent antifungal peptide pathway. The development of invasive hyphae by C. tropicale owing to the reduction of preinvasive resistance then triggers the blockage of further hyphal expansion via the activation of the second layer of resistance, i.e., postinvasive resistance, which guarantees the robustness of the nonhost resistance of A. thaliana against Colletotrichum pathogens. Both the tryptophan-derived metabolic pathway and glutathione synthesis play critical roles in the postinvasive resistance against C. tropicale, although the molecular mechanism of postinvasive resistance remains to be elucidated. In this review, we describe the current understanding of the molecular background of the Arabidopsis nonhost resistance against Colletotrichum fungi and discuss perspectives for future research on this durable resistance.     

Antifungal activity of a novel compound purified from the culture filtrate of <Emphasis Type="Italic">Biscogniauxia</Emphasis> sp. O821 against the rice blast fungus <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

Rice blast is a devastating fungal disease resulting in major losses to rice crops. Owing to continuous acquisition of resistance by the causal fungus, several fungicide chemicals are no longer effective. Therefore, there is a need to identify natural components and develop new agents to control fungal pathogens. We previously demonstrated that the culture filtrate of Biscogniauxia sp. O821 inhibited infection behavior of Magnaporthe oryzae and subsequent blast lesion formation. In the present study, we isolated a new compound, (3aS,4aR,8aS,9aR)-3a-hydroxy-8a-methyl-3,5-dimethylenedecahydronaphto[2,3-b]furan-2(3H)-one (HDFO), from the culture filtrate of Biscogniauxia sp. O821 and determined its molecular weight as 248. The HDFO structure was determined by electrospray ionization-mass spectrometry and nuclear magnetic resonance spectroscopy after purification with column chromatography and high-performance liquid chromatography. The structure of this antifungal compound was similar to that of alantolactone and isoalantolactone. The growth inhibition zone against M. oryzae in presence of HDFO was observed at Rf 0.5–0.6 on a thin layer chromatography plate. HDFO inhibited conidial germination of M. oryzae in a dose-dependent manner (1–200 ppm). Furthermore, blast lesion formation was significantly suppressed by HDFO at over 5 ppm. These results suggest that HDFO from the culture filtrate of Biscogniauxia sp. O821 can protect rice from rice blast disease caused by M. oryzae. This is the first report that HDFO produced by Biscogniauxia sp. can serve as an antifungal compound against M. oryzae.     

Potential use of Eucalyptus globulus essential oil against Phytophthora colocasiae the causal agent of taro leaf blight

This study was carried out to determine the antimicrobial activity of essential oil derived from leaves of eucalyptus (Eucalyptus globulus) against Phytophthora colocasiae, the causal agent of taro leaf blight (TLB). Essential oil was obtained by Clevenger-type water distillation. The major compounds in this essential oil were identified using gas chromatography (GC) and gas chromatography coupled with mass spectrometry (GC/MS). Antimicrobial activity of the essential oil was tested in vitro against mycelial growth, sporangia and zoospores germination of P. colocasiae. Additionally, in situ tests were conducted on detached healthy taro leaves discs and evaluating necrosis symptoms of TLB were assessed. Results of the chemical composition of the essential oil analysis showed that, 1.8-cineole (26.4 %), α-pinene (14.1 %) and p-cymene (10.2 %) are the most abundant compounds. In addition, four components could be identified in noticeable amounts (18.1 % in the total): regulator G1/G2 and its derivatives as well as flavesone, a regulator G3 derivative. G is a generic term for a family of 2,3-dioxabicyclo[4,4,0] decane system growth regulators. Here, G-regulators and derivatives are described for the first time in E. globulus. Antimicrobial activity of the essential oil was recorded with total inhibition of mycelia growth and sporangia germination at 0.625 mg/ml, while the complete inhibition of zoospores germination was recorded at 0.156 mg/ml. In situ results showed that essential oil completely inhibited the appearance of disease symptoms, necrosis development and sporulation at 3.5 mg/ml. These results demonstrated that, the essential oil of Eucalyptus globulus has high ability to inhibit the development of P. colocasiae, and might be used for controlling TLB.     

Effect of mint oil against <Emphasis Type="Italic">Botrytis cinerea</Emphasis> on table grapes and its possible mechanism of action

This paper assessed the antifungal effects of sage oil, lavender oil, mint oil, and tea tree oil on the postharvest fungal pathogen, Botrytis cinerea, which causes gray molds. The change of morphology, physiological, and biochemical characteristics about fungal hyphae and conidia were determined. As results show, all four oils can effectively inhibit the growth of B.cinerea and the antifungal effects are dose dependent. The best antifungal effect has been found from mint oil. According to in vitro studies, volatile vapor worked better than direct contact. With volatile vapor, the growth of B.cinerea was inhibited completely at 2 for all four oils. Mint oil at 500 μL/L and its volatile vapor at 25 could inhibit both conidia germination and disease incidence significantly in vivo. Observations by using scanning electron microscope and transmission electron microscope revealed that, mint oil could destroy the ultrastructure of hyphae and conidia, resulting in markedly shriveling and crinkling of the hyphae and conidia. It could also thicken and disrupt cell wall, causing cellular nucleic acids and proteins divulged with the damage of the cell wall. The chemical composition analysis of mint oil using GC/MS revealed that its main components were cyclohexanol, cyclohexanone, and some alkenes and alkanes. The majority of the components were effective antifungal agents. The content of volatile cyclohexanol and cyclohexanone were found to be 39.79% and 22.24% respectively.     

Integrative diagnosis and molecular phylogeny of dagger and needle nematodes of olives and grapevines in the island of Crete,Greece, with description of Xiphinema cretense n. sp. (Nematoda,Longidoridae)

The occurrence and geographic distribution of longidorid nematode species inhabiting the rhizosphere of cultivated and wild olive and grapevine in Crete Island were investigated. Morphological and morphometrical studies identified five Longidorus and six Xiphinema species, with frequencies of prevalence (for wild and cultivated olives and grapevines, respectively) as follows: Longidorus closelongatus (2.0–13.3 %), L. cretensis (1.0–6.7 %), L. moesicus (13.3 % only in grapevines), L. orientalis (3.3 % only in grapevines), L. pseudoelongatus (7.0 % only in olives), Xiphinema cretense n. sp. (3.0 % only in olives), X. index (3.0–23.3 %), X. israeliae (6.3 % only in olives), X. italiae (3.3–10.0 %), X. pachtaicum (26.7–42 %) and X. simile (3.3 % only in grapevines). Xiphinema cretense n. sp. is characterized by a body size 3,872–6,135 μm long, lip region anteriorly rounded, separated from the rest of the body by a depression, odontostyle and odontophore 140.6 and 80.3 μm long respectively, vulva position at 46.0–50.5 %, female tail 31.0–38.0 μm long, nearly hemispherical with curvature essentially dorsal and with a tip completely rounded or presenting a very short bulge, c ratio (119.1–186.9), c’ ratio (0.7–0.8). Molecular characterisation using D2-D3 expansion regions of 28S rRNA, 18S rRNA and ITS1-rRNA was carried out and maximum likelihood and Bayesian inference analysis were used to reconstruct phylogenetic relationships among these species and with other longidorids.     

Evaluation of Gram-positive rhizosphere and endophytic bacteria for biological control of fungal rice (<Emphasis Type="Italic">Oryzia sativa</Emphasis> L.) pathogens

Gram-positive bacteria isolated from the rhizosphere and inside the roots of rice were characterized for plant growth promoting (PGP) traits and antifungal activity against some rice plant pathogenic fungi of rice. The results showed the endophytic and rhizosphere isolates had different PGP traits and antifungal activity. Only one rhizosphere isolate and one endophytic isolate showed highly inhibitory effects against the mycelial growth of all fungal rice pathogens tested in this study. The best bacterial isolates, based on multiple PGP traits and inhibitory effects against the mycelial growth of all fungal rice pathogens, were identified. Based on biochemical tests and by comparison of 16S rDNA sequences, the endophytic isolate REN₃ and the rhizosphere isolate REN₄ were closely related to Bacillus cereus and Bacillus mojavensis respectively. The broad-spectrum antifungal strains, the REN₃ and REN₄ isolates analyzed here, exert multiple PGP and antagonistic activity and represent an excellent option to be used as either potent bio-promoting or bio-control agents in rice under in vitro conditions. This application may help to minimize dependence on pesticides, which have adverse effects on the environment, finally leading to have sustainable environments. In conclusion, the results of antifungal activity showed rice harbors bacteria with a good potential in biocontrol of rice fungal pathogens.     

A rapid and miniaturized system using Alamar blue to assess fungal spore viability: implications for biosecurity

Long-lasting viable fungal spores are one of the important aspects in emergence, spread and disease development of pathogenic fungi. We developed a rapid and miniaturized system using Alamar Blue (resazurin dye; 7-hydroxy-3H-phenoxazin-3-one 10-oxide) for assessing fungal spore viability, using the ascomycete Leptosphaeria maculans (causing blackleg disease on canola) as a ‘model pathogen’. The assay is dependent on the metabolic activity of viable fungal spores to convert the dark blue of resazurin (maximum absorbance 605 nm) into the pink colour of resorufin (maximum absorbance 573 nm). The Alamar Blue assay uses an optimised micro-titre based format that was far superior for determining fungal spore viability in comparison with current conventional techniques including trypan blue staining, a TC10 cellometer cell counter, or by assessing germination of the spores under the microscope. This new assay was also more rapid and reproducible than current conventional tests to detect viable spores. Viable spores could be reliably detected within two hours. The successful application of the Alamar Blue assay to measure fungal spore viability in the current study has important benefits for biosecurity operations relating to faster and more reliable confirmation of viability of potential invasive exotic fungal pathogens and in minimising any consequent disease outbreaks. The effectiveness of the Alamar Blue assay was confirmed by successfully determining the relative retention times of viable L. maculans ascospores across a range of different potential spore-carrier materials, including steel, fabric, wood, paper, rubber and leather, over a time period of eight months. To further confirm the wide applicability of the Alamar Blue assay, it was successfully applied to detect viable spores of fungal pathogens of diverse taxonomic groups, including Kabatiella caulivora, Magnaporthe oryzae and Puccinia striiformis f.sp. tritici, and also of the yeast Saccharomyces cerevisiae.     

Plant ferredoxin-like protein enhances resistance to bacterial soft rot disease through PAMP-triggered immunity in Arabidopsis thaliana

The protection of plants against bacterial disease is one of the important issues that need to be studied in agricultural applications. The application of a transgene, such as a gene that encodes plant ferredoxin-like protein (PFLP), to generate resistant plants is one possible strategy. Our previous reports have demonstrated that transgenic plants that express extracellular PFLP (ESF plants) are more resistant to bacterial pathogens. This protein intensifies the hypersensitive response (HR) in plants when they are infiltrated by a pathogen-associated molecular pattern (PAMP), harpin (HrpZ), from Pseudomonas syringae. In addition, this intensified HR is associated with the expression of membrane-bound NADPH oxidase. Thus, we attempted to determine the involvement of PFLP in intensifying PAMP-triggered immunity (PTI) to enhance disease resistance. First, we showed that transgenic Arabidopsis plants with the pflp gene were resistant to bacterial soft rot caused by Pectobacterium carotovorum subsp. carotovorum (Pcc). Then, the fliC gene which encoded flagellin from Pcc was cloned and expressed. The FliC protein was used in the functional study with PFLP in Arabidopsis Col-0 plants. The reactive oxygen species (ROS) generation and HR ratio were induced by the treatment with both PFLP and FliC together, but they were not induced by treatment with PFLP or FliC alone. Similar results were confirmed in ESF plants, where FliC elicited rapid ROS accumulation and callose deposition. Moreover, we demonstrated that the PFLP-intensified ROS generation and HR were related to Ca²⁺ influx and activation of NADPH oxidase. We concluded that the PFLP-intensified disease resistance is associated with the intensification of PAMP-triggered immunity.     

Development of conventional and real-time PCR protocols for specific and sensitive detection of Colletotrichum capsici in chilli (Capsicum annuum L.)

Anthracnose, caused by Colletotrichum capsici, is a major disease of chilli (Capsicum annuum L.) affecting both fruit and seed quality. The pathogen is both internally and externally seedborne. However, a rapid and sensitive method for detection of this pathogen in seeds is currently limited. In this study, a polymerase chain reaction (PCR) method based on sequence characterized amplified region (SCAR) marker was developed for specific and sensitive detection of C. capsici in chilli seeds and fruits. The developed SCAR primers were highly specific to C. capsici and resulted in the amplification of an expected 250-bp fragment from genomic DNA of all seven of the C. capsici isolates tested. No amplification occurred when the SCAR primers were tested with genomic DNA from three other fungal isolates and four other Colletotrichum species. The SCAR primers successfully amplified similar sized fragments from DNA derived from C. capsici-infected chilli fruits. The molecular detection sensitivity of C. capsici was 1 pg of purified C. capsici DNA template and 25 ng of DNA from C. capsici-infected chilli fruits. A real-time PCR assay was also developed using SYBR Green chemistry for detection of C. capsici in chilli fruits and seeds. The standard curve obtained showed a linear correlation between copy number of the cloned target DNA sequence of C. capsici and cycle threshold (Ct) values, with R² of 0.98. These PCR-based assays may be highly useful in detection of this important pathogen in chilli seeds and fruits in plant quarantine laboratories.     

In vitro-Untersuchung einer möglichen fungistatischen Wirkung des Sekrets der Weinbergschnecke (Helix pomatia,Helix aspersa) auf Botrytis cinerea

In viticulture practices, technical cultivation practices and application of fungicides, specifically named botryticides, are important to control the grey mold, Botrytis cinerea. While conventional disease control is based upon applications of fungicides, options for controlling of grey mold in organic viticulture is limited. Resistance of the fungus and multiple drug resistance (MDR) require development of new disease control strategies. In vivo observation showed a reduced grey mold infection of grapes if covered with slime from garden snail. Hence, snail slime, commercial lectin (Helix aspersa agglutinin, HAA) from garden snail and bacteria isolated from snail slime were tested for antifungal or antagonistic activities against B. cinereain vitro. Furthermore, fluorescein isothiocyanate labeled Helix-lectin (FITC-HPA) was used to detect terminal N?acetyl-galactosamine residues (GalNAc) on fungal cell wall surface. In our experiments, neither slime (after sterile filtration) nor commercial lectin nor slime bacteria were found to affect spore germination and mycelial growth. Binding of lectin was found to depend on media and age of the fungal mycelium.     

Molecular variability of Apple chlorotic leaf spot virus in Shaanxi,China

Apple chlorotic leaf spot virus (ACLSV) is one of the latent viruses that occur in apple orchards worldwide but usually without visible symptoms. In 2010–2012, a total of 550 apple leaf samples from 12 different major apple-producing areas in Shaanxi, China, were tested by serological assay for ACLSV; the results revealed an infection level of 51.5 %. Because of the known variability in the putative amino acid sequences of the coat protein (CP), and thus the potential for non-detection by serological assay, the molecular variability of isolates of ACLSV collected in Shaanxi was analyzed using PCR and compared with isolates from the rest of the world. Sequences of 504 nt corresponding to 87 % of the CP gene of 12 isolates were acquired by RT-PCR and deposited in GenBank with the accession numbers KF134387–KF134298. Comparisons of the partial CP gene sequences of these 12 isolates as well as isolates previously reported in the world revealed the pairwise identities ranging from 68.9–99.8 % and 73.8–100 % at the nucleotide and amino acid level, respectively. Phylogenetic analysis based on these nucleotide sequences showed that the 72 isolates deposited in GenBank fell into three groups (P205, B6 and Ta Tao 5 Group). Our 12 ACLSV isolates were separated into the P205 and B6 groups, respectively. Multiple alignment analysis of the amino acid sequences of CP revealed that there was a combination of six amino acids at positions 40, 59, 75, 86, 130 and 184 in isolates from each group that could be used to distinguish among the three groups. Two recombination events were identified from all isolates by recombination analysis, and three ACLSV isolates collected in this study participated in these two events. Our results show that molecular variation was present in isolates of ACLSV collected in Shaanxi province and this may reflect introductions of the virus associated with different sources of germplasm.     

Analysis of genetic and virulence variability of Stemphylium lycopersici associated with leaf spot of vegetable crops

Stemphylium lycopersici (Enjoji) W. Yamam was initially described from tomato and has been reported to infect different hosts worldwide. Sequence analyses of the internal transcribed spacer (ITS) regions 1 and 2, including 5.8S rDNA (ITS-5.8S rDNA) and glyceraldehyde-3-phosphate dehydrogenase (gpd) gene, random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR), as well as virulence studies were conducted to analyze 46?S. lycopersici isolates. Stemphylium lycopersici isolates used in this study were obtained from diseased tomato (Solanum lycopersicum L.), eggplant (Solanum melongena L.), pepper (Capsicum annuum L.) and lettuce (Lactuca sativa L.) from major vegetable growing regions of Malaysia, including the three states of Pahang, Johor and Selangor between 2011 and 2012. Phylogenetic analysis of a combined dataset of the ITS-5.8S rDNA and gpd regions indicated that all isolates were clustered in the sub-cluster that comprised S. lycopersici, and were distinguished from other Stemphylium species. Cluster analyses using the UPGMA method for both RAPD and ISSR markers grouped S. lycopersici isolates into three main clusters with similarity index values of 67 and 68 %. The genetic diversity data confirmed that isolates of S. lycopersici are in concordance to host plants, and not geographical origin of the isolates. All S. lycopersici isolates were pathogenic on their original host plants and showed leaf spot symptoms; however, virulence variability was observed among the isolates. In cross-inoculation assays, the representative isolates were able to cause leaf spot symptoms on eggplant, pepper, lettuce and tomato, but not on cabbage.     

Identification and species status of the mango biotype of Colletotrichum gloeosporioides in Ghana

Research work was carried out to identify and ascertain the species status of the mango biotype of Colletotrichum gloeosporioides infecting mangoes in Ghana. Forty five isolates of Colletotrichum species were collected from 12 districts in Ghana while five each were obtained from mango fruits from Florida, Mexico and Puerto Rico. The entire internal transcribed spacer region, partial beta-tubulin gene and partial glyceraldehyde-3-phosphate dehydrogenase gene of isolates were sequenced and used in phylogenetic studies. The results of the sequence analysis of the first ribosomal transcribed spacer (ITS 1) region showed that 35 % of the isolates from Ghana and all the five isolates from Mexico were the mango biotype of C. gloeosporioides, while the others were not. Phylogenetic studies showed that the mango biotype of the pathogen was Colletotrichum asianum but not C. gloeosporioides as previously thought. However, the other isolates that were not the mango biotype were identified as Colletotrichum siamense and Colletotrichum species which had probably cross-infected mango from other fruit crops in the field.     

Detection and occurrence of melon yellow spot virus in Ecuador: an emerging threat to cucurbit production in the region

More than fifty viruses have been reported in cucurbit crops worldwide. In Ecuador, approximately 3,000 ha of watermelon, melon and cucumbers are cultivated annually, but there have been few studies to identify viruses responsible for epidemics. During this study, sequencing of double-stranded RNA (dsRNA) extracted from watermelon and melon leaves showing virus-like symptoms revealed the presence of Melon yellow spot virus (MYSV, genus Tospovirus) and the partially described Cucumis melo endornavirus (CmEV). Specific primers, designed to detect each virus, showed that MYSV was present in 40, 64 and 67 % of symptomatic watermelon, cucumber and melon samples, respectively. For CmEV, 95 % of both symptomatic and asymptomatic melon plants tested positive. However, the virus was not detected in watermelon or cucumber. Sequence comparisons showed nucleotide identities of 97 % and 94 % for the polymerase and the nucleocapsid protein, respectively, between the Ecuadorean MYSV and the one reported from Japan. To the best of our knowledge, this is the first report of MYSV and CmEV in Ecuador and the Americas.     

A SCAR marker specific for rapid detection of the avirulence gene Avr1c in Phytophthora sojae

The F₂ population derived from a cross between isolates pR_x (Avr1c-Avr1c) and ps₁ (avr1c-avr1c) of Phytophthora sojae, fungal agent of soybean stem and root rot, was used to determine the genetic basis of avirulence towards Rps1c gene in soybean. The results indicated that this avirulence is dominant and controlled by a single locus, as expected for a simple gene-for-gene model. Segregation of Avr1c in the F₂ progeny of this cross fits a 3:1 ratio. Four of 80 AFLP primers effectively distinguished the avirulent pR_x from the virulent ps₁. Among the 5 specific markers, band C was amplified from the avirulent pR_x by primer set EGC/MAT, then recovered and cloned. This AFLP marker was successfully transfered to a SCAR marker through sequencing, primer design and specific amplication of the DNA of the avirulent pR_x. Results of validity and specificity experiments with 50 individuals of the F₂ progeny and 50 field isolates demonstrated that this SCAR marker (a 616-bp fragment) can be successfully and specifically amplified from the P. sojae isolates that have Avr1c gene.     

Direct antifungal activity of tiadinil,a systemic acquired resistance inducer,and thymol formulations on <Emphasis Type="Italic">Stagonosporopsis citrulli</Emphasis> and control of watermelon gummy stem blight

This study evaluated the direct antifungal activity of tiadinil [N-(3-chloro-4-methylphenyl)-4-methyl-1,2,3-thiadiazole-5-carboxamide], a systemic acquired resistance (SAR) inducer and two formulations of thymol (thymol I and thymol II) against Stagonosporopsis citrulli, the causal agent of gummy stem blight (GSB) disease of watermelon. Tiadinil, thymol I and thymol II completely inhibited the mycelial growth of S. citrulli in vitro at ≥?100 ppm. Conidial germination and germ tube elongation were completely inhibited by tiadinil at ≥?2000 ppm and by thymol-based formulations at ≥?100 ppm. A single foliar application of tiadinil at ≥?10 ppm or a single application of thymol I and II at ≥?1 ppm, 48 h before or after pathogen inoculation, significantly reduced disease severity of watermelon seedlings inoculated with 10⁵/ml conidial suspension of S. citrulli, compared to respective nontreated controls. Plants treated with foliar application of tiadinil at ≥?1000 ppm before pathogen inoculation had significantly lower disease severity than plants that received an equivalent drench application. The efficacy of foliar application of tiadinil was affected by concentration and frequency of application. The study suggests direct antifungal activity of tiadinil, indicating a new mode of action of tiadinil against GSB disease of watermelon. The study also demonstrated direct antifungal action of thymol, a formulated active compound of essential oils, against S. citrulli and GSB disease of watermelon.