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.     

Identification and characterization of <Emphasis Type="Italic">Colletotrichum</Emphasis> spp. associated with chili anthracnose in peninsular Malaysia

Anthracnose fruit rot caused by Colletotrichum spp. is a serious post-harvest disease of chili fruits (Capsicum spp.). One hundred-thirty isolates of Colletotrichum spp. were isolated from anthracnose of green and red cayenne pepper (Capsicum annuum) and bird’s eye chili (Capsicum frutescens). The isolates were morphologically identified as Colletotrichum acutatum sensu lato (62 isolates), Colletotrichum truncatum (54 isolates), and Colletotrichum gloeosporioides sensu lato (14 isolates). Molecular identification and phylogenetic analyses were based on internal transcribed spacer regions, β-tubulin, actin, and glyceraldehyde-3-phosphate dehydrogenase genes, and the isolates were re-identified as C. scovillei (58 isolates), C. truncatum (54 isolates), C. siamense (11 isolates), C. fioriniae (four isolates), and C. fructicola (3 isolates). Maximum likelihood trees using combined sequences showed that isolates of the same species grouped in the same main clade with the isolates used for comparison. Pathogenicity testing showed that the tested isolates from each species were pathogenic towards green and red Capsicum annuum and Capsicum frutescens upon treatment of wounded fruit, using both the mycelial plug and conidial suspension methods. Only five isolates of C. truncatum and seven isolates of C. scovillei were found to be pathogenic upon treatment of unwounded fruit. The occurrence of five Colletotrichum spp. (C. siamense, C. fructicola, C. scovillei, C. fioriniae, and C. truncatum) associated with chili anthracnose in Peninsular Malaysia indicates that correct species identification is important to formulate not only effective disease management, but also effective quarantine policy.     

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.     

A loop mediated isothermal amplification (LAMP) assay for rapid and reliable detection of <Emphasis Type="Italic">Anguina wevelli</Emphasis>, a grass parasitic nematode

Anguina wevelli is a pathogenic grass parasitic nematode, however it is difficult to identify based simply on morphology. This study developed a loop-mediated isothermal amplification (LAMP) assay to detect A. wevelli. The LAMP method developed could specifically detect A. wevelli in 45 min, and the detection limit was 1/80000 of the total DNA extracted from a single juvenile (J2), an equivalent of 2.5 pg of DNA. This is the first report of the detection of Anguina spp. by using a LAMP-based method.     

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.     

Development of a loop-mediated isothermal amplification assay for sensitive and specific detection of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">cucumerinum</Emphasis> Owen

Fusarium wilt, caused by Fusarium oxysporum f. sp. cucumerinum Owen (FOC), is a destructive disease affecting cucumber production worldwide. Developing an accurate and reliable method for detection of FOC is important for disease prediction and control. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed and validated for specific and sensitive detection of FOC. Four LAMP primers were designed based on the sequence of the FOC-specific random amplified polymorphic DNA (RAPD) marker OPZ-12₈₆₅. LAMP reactions were performed at different temperatures and for different durations, and the optimal temperature and duration were 63 °C for 60 min, respectively. Hence, a LAMP assay for detection of FOC was established. The specificity of the LAMP method was evaluated against 119 isolates of FOC and other pathogens, and only FOC isolates yielded positive results. In sensitivity tests, the lowest concentration of genomic DNA required for the LAMP assay was 10 fg in a 25 μL reaction. The LAMP assay was successfully applied to detect FOC in cucumber tissues and soil from infested fields, and the positive ratios of LAMP, PCR, and traditional tissue isolation for detecting FOC from diseased cucumber root samples were100%, 86.6 and 83.3%, respectively. Therefore, the LAMP assay developed herein should serve as a simple, cost-effective, rapid, highly specific, and sensitive tool for the visual detection of FOC and contribute to improved disease management.     

Markers to differentiate species of anthracnose fungi identify <Emphasis Type="Italic">Colletotrichum fructicola</Emphasis> as the predominant virulent species in strawberry plants in Chiba Prefecture of Japan

Colletotrichum fungi belonging to the Colletotrichum gloeosporioides species complex include a number of economically important postharvest pathogens that often cause anthracnose. Until now, different species within this group could only be distinguished from one another reliably using multigenic phylogenetic analyses. Using a comparative genomics approach, we developed a marker that can differentiate Colletotrichum fructicola, Colletotrichum aenigma and Colletotrichum siamense within the C. gloeosporioides species complex based on PCR amplicon size differences. When we used this marker to classify 115 isolates collected over 20 years from strawberries in Chiba Prefecture, Japan, the isolates were predominantly C. fructicola. To our knowledge, this is the first report characterizing different species of Colletotrichum infecting strawberries in Japan and contributes to our understanding on the diversity of anthracnose pathogens in Japan.     

Role of secondary metabolites in the biocontrol activity of <Emphasis Type="Italic">Pseudomonas corrugata</Emphasis> and <Emphasis Type="Italic">Pseudomonas mediterranea</Emphasis>

Colletotrichum gloeosporioides is the causal agent of Camellia oleifera anthracnose, mainly infecting fruits and leaves. The fungus secretes degrading enzymes to destroy the cuticle of aerial plant parts and help infect the host successfully. To validate whether a cutinase gene (CglCUT1) was required for cutinase activity and pathogenicity of C. gloeosporioides, the CglCUT1 gene was cloned and analyzed. The characterization of CglCUT1 predicted protein suggests that the cloned DNA encoded a cutinase in C. gloeosporioides affecting C. oleifera. The CglCUT1 showed a high homology to those from C. gloeosporioides causing papaya anthracnose and C. capsici causing pepper anthracnose, as well as those of other ascomycetes. The whole CglCUT1 gene was knocked-out and the knockout mutant (?CglCUT39) was subsequently complemented using Agrobacterium tumefaciens mediated transformation. The knockout transformants exhibited significant decreases in cutinase activity and virulence compared with the wild-type strain. The complemented transformants of the disrupted transformant ?CglCUT39 showed a significant increase in cutinase activity and virulence compared with the disrupted transformant ?CglCUT39. This study suggests that the CglCUT1 gene has a positive effect on fungal virulence of the hemibiotrophic C. gloeosporioides on C. oleifera.     

The potential of fludioxonil for anthracnose control on China chili fruit

Anthracnose, caused by Colletotrichum scovillei, is one of the most destructive disease which causes massive yield losses in chili. Our preliminary study has demonstrated that fludioxonil had high activity against mycelial growth and spore germination of C. scovillei but it has not been labeled on chili. The aim of this study was to investigate the impact of fludioxonil on C. scovillei infection. In greenhouse and field trials, fludioxonil was applied to chili plants to determine the duration of protection of fruit provided by this compound against chili anthracnose. Fludioxonil may have impact on the early stage of chili anthracnose infection by inhibiting the germ tube elongation and appressorium formation. Moreover, it inhibited the secondary infection of C. scovillei by inhibiting the sporulation, germination of spores and formation of appressoria. Fludioxonil provided high level of protective activity for up to 96 h, as well as excellent curative activity of within 24 h in vivo tests. In field trials, fludioxonil can reduce the incidence and severity of chili anthracnose while giving a higher chili yield. These results suggest that fludioxonil could be a promising fungicide for anthracnose control in chili production.     

Assessment of the baseline sensitivity and resistance risk of <Emphasis Type="Italic">Colletotrichum acutatum</Emphasis> to fludioxonil

Anthracnose, which is caused by Colletotrichum acutatum, is a destructive disease of pepper. A preliminary study demonstrated that fludioxonil (a phenylpyrrole fungicide) has good activity against C. acutatum and thus has potential to be used as an alternative fungicide for the management of pepper anthracnose. However, there is no information regarding the baseline sensitivity and resistance risk of C. acutatum to fludioxonil. Thus, the sensitivities of 205 isolates of C. acutatum to fludioxonil were determined. The results showed that the frequency distributions of the EC₅₀ values were unimodal, and the mean EC₅₀ values for the inhibition of mycelial growth and spore germination were 0.031 μg/mL and 0.035 μg/mL, respectively. Three stable mutants with high resistance to fludioxonil were obtained in the laboratory. Two parameters, namely in vitro sporulation and the in vitro and in vivo germination of spores, showed significant difference (P < 0.01) when the mutants were compared to the sensitive isolates. Moreover, the mutants were more sensitive to osmotic stress compared to the parents. No significant differences (P ≥ 0.05) were detected in colony diameter, mycelia weight, pathogenicity or sporulation in vivo between the fludioxonil-resistant mutants and their corresponding parents. Cross-resistance occurred between fludioxonil, iprodione and procymidone. Overall, resistance risk of C. acutatum to fludioxonil was low to medium, and thus resistance management should be considered.     

Molecular and phenotypic characterization revealed six <Emphasis Type="Italic">Colletotrichum</Emphasis> species responsible for anthracnose disease of small cardamom in South India

Small cardamom (Elettaria cardamomum Maton) is extensively cultivated in the Western Ghats of South India either as a monocrop under the forest trees or as an intercrop along with arecanut and coffee plantations. Colletotrichum species responsible for severe outbreaks of anthracnose on small cardamom in South India are reported. Small cardamom anthracnose, popularly known as “Chenthal”, manifests itself on the foliage as yellowish lesions, which later coalesce to form large blighted areas. In advanced stages, the affected leaves dry up giving a burnt appearance to the plant. Twenty-five isolates of Colletotrichum were isolated from leaves of small cardamom in Karnataka, Kerala and Tamil Nadu states of India. The isolates were characterized through morphological studies and multilocus phylogenetic analysis (ITS, ACT, CHS-1, GAPDH, TUB2, CYLH3, GS and ApMat gene regions) to test whether different species are present and identified: C. karstii (2 isolates), C. gloeosporioides (1), C. siamense (7), C. syzygicola (6), Colletotrichum sp (5), and C. guajavae (4), as the cause of anthracnose on small cardamom for the first time. Pathogenicity of the six species was confirmed. To our knowledge, this is the first detailed study of Colletotrichum species which cause anthracnose diseases on small cardamom.     

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

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

Subcelullar localization of proteins associated with <Emphasis Type="Italic">Prune dwarf virus</Emphasis> replication

The fungus Colletotrichum lindemuthianum is the causal agent of anthracnose, one of the most severe diseases of the common bean (Phaseolus vulgaris). The infection process begins with the adhesion of conidia to the plant’s surface. Appressoria are then formed, allowing penetration of the fungus. Next, the biotrophic phase begins, followed by the necrotrophic phase. Due to the peculiar nutrition mode of the fungus, including both of the previously mentioned stages, it is of great interest to determine which genes are involved in the transition between the two phases during the infection process. To determine this, suppression subtractive hybridization (SSH) was used in association with qRT-PCR in the present study. These methods allowed for the identification of genes that were differentially expressed at each developmental stage of the fungus in the plant. This is the first report on the use of the cited techniques to evaluate the infectious cycle of the fungus. A total of 175 sequences exhibited significant identity (e?≤?10^?5) with sequences present in the sequenced genomes of P. vulgaris and C. lindemuthianum; approximately 41 % of those were determined to belong to the fungus, and 59 % were determined to belong to the plant. Of the predicted sequences, 68 % were of unknown function or were not found in the databases. Among the analyzed expressed sequence tags (ESTs), sequences were found that encode proteins related to: primary and secondary metabolism; the transport of different compounds; the degradation/modification of proteins; cell regulation and signaling; cellular stress response; and the degradation of exogenous compounds. The obtained results allowed for the identification of sequences encoding proteins that are essential for the progression of anthracnose. Furthermore, it was possible to identify new genes, the functions of which have not yet been described, and even to identify unique genes of C. lindemuthianum that are involved in the pathogenicity and virulence of this fungus.     

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.     

Practical method combining loop-mediated isothermal amplification and bait trap to detect <Emphasis Type="Italic">Pythium helicoides</Emphasis> from hydroponic culture solutions

Two detection methods combining loop-mediated isothermal amplification (LAMP) and a bait trap were developed to detect Pythium helicoides in greenhouses containing roses, miniature roses, and poinsettias in hydroponic culture systems. In “Bait-LAMP”, a crude extract derived from perilla seeds as the bait was used in the LAMP reaction, whereas in the “Bait culture-LAMP”, a crude extract of mycelia grown out from perilla seeds onto Pythium-selective medium served as the bait. The two methods are simple and rapid for practical monitoring of P. helicoides in hydroponic culture systems.     

A myosin passenger protein gene (<Emphasis Type="Italic">FaSmy1</Emphasis>) is an essential regulator of cell development,pathogenicity, DON biosynthesis,and resistance to the fungicide phenamacril in <Emphasis Type="Italic">Fusarium asiaticum</Emphasis>

Wheat streak mosaic virus (WSMV) and Triticum mosaic virus (TriMV) are important viruses of wheat (Triticum aestivum L.) in the Great Plains of United States. In addition to agronomic practices to prevent damage from these viruses, temperature sensitive resistance genes Wsm1, Wsm2 and Wsm3, have been identified. However, threshold temperatures for Wsm1 and Wsm3 have not been clearly defined. To better understand these two resistance genes, wheat lines C.I.15092 (Wsm1), KS96HW10–3 (Wsm1), and KS12WGGRC59 (Wsm3) were evaluated for WSMV resistance at 27, 30, 33 and 35 °C and for TriMV resistance at 18, 21, 24, 27, 30, 33 and 35 °C. The results showed that only C.I.15092 remained resistant at 30 °C for both viruses. This line also tolerated TriMV at 33 and 35 °C with less sever symptom and lower infection rates. Wheat lines KS96HW10–3 and KS12WGGRC59 hold resistance to TriMV up to 21 °C. Molecular marker results suggested that the resistance in C.I.15092 is most probably conditioned by the resistance gene Wsm1 and additional gene(s) other than Wsm2 and Wsm3.     

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.     

Rapid and sensitive detection of <Emphasis Type="Italic">Meloidogyne mali</Emphasis> by loop-mediated isothermal amplification combined with a lateral flow dipstick

Meloidogyne mali Itoh, Ohshima & Ichinohe, 1969 is a polyphagous root-knot nematode that infects a wide range of host plants and is subject to phytosanitary restrictions in many countries, including China. In this study, we integrated the loop-mediated isothermal amplification (LAMP) technique with visual detection by a lateral flow dipstick (LFD) and developed an accelerated LAMP-LFD method for the rapid identification of M. mali, targeting the internal transcribed spacer regions and 5.8S ribosomal DNA sequence (ITS-5.8S). The entire test could be performed within 45 min, including 35-min LAMP amplification, 5-min hybridization, and 3–5-min visualization on LFD. The detection limit of this method was 2-pg bulked M. mali gDNA per reaction, and the equivalent of 1‰ single female adult or 1% single second-stage juvenile (J2) could be detected. It is recommended that DNA is extracted from single J2 isolated from soil samples by the modified Baermann funnel method or from an adult female from galls. Based on this, the field sensitivity and specificity of this LAMP-LFD method for the detection of M. mali in field soil samples were both 100% compared to traditional microscopic observation. These results indicate that the LAMP-LFD method is rapid, sensitive, accurate, and simple, and would be applicable for the routine monitoring of phytosanitary M. mali.     

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.     

Quantitative detection of <Emphasis Type="Italic">Colletotrichum godetiae</Emphasis> and <Emphasis Type="Italic">C. acutatum sensu stricto</Emphasis> in the phyllosphere and carposphere of olive during four phenological phases

A duplex qPCR detection method was developed to detect and quantify Colletotrichum godetiae and C. acutatum sensu stricto (s.s.) in olive tissues. The method proved highly specific and sensitive with a detection limit of 10 pg for each pathogen. The analysis of green and senescent leaves, fertilized fruitlets with floral residues, green fruit and symptomatic and asymptomatic fruit collected in May, June, October and December revealed a high incidence of both C. godetiae and C. acutatum s.s. in Calabria, southern Italy. In comparison with previous reports, these results highlighted an ongoing population shift from C. godetiae to C. acutatum s.s. Interestingly, C. godetiae was slightly more abundant in terms of number of infected samples, yet the quantity of C. acutatum in infected samples was always higher, suggesting greater aggressiveness and/or sporulation ability of the latter pathogen. The populations of both C. godetiae and C. acutatum s.s. increased sharply in December even though both pathogens were detected widely in asymptomatic samples in May, June and October, confirming an important role of latent infections in the disease cycle. A large quantity of both C. godetiae (1.7 × 10⁸ cells/mg of tissue) and C. acutatum s.s. (7.5 × 10⁸ cells/mg of tissue) was estimated in symptomatic fruit, presenting an enormous inoculum potential for secondary infections. Two other important observations were a high incidence and quantity of both pathogens in senescent leaves and in fertilized fruitlets with floral residues as compared to green leaves.