Assessment of the effect of surface drip irrigation on Verticillium dahliae propagules differing in persistence in soil and on verticillium wilt of olive

For efficient integrated management of verticillium wilt in olive (VWO), it is important to establish whether irrigation treatments (with Verticillium dahliae‐free water) that mitigate the disease in V. dahliae‐infested soil, also reduce the levels of more and less persistent propagules of the pathogen in the soil. Effects of irrigation on VWO and V. dahliae propagules were evaluated under natural environmental conditions. Potted plants were irrigated (pathogen‐free water) to two ranges of soil water content (RWC; high and low) at three surface drip‐irrigation frequencies (daily, weekly, and daily during some periods and otherwise weekly). VWO and total inoculum density (ID), density of less persistent micropropagules (MpD) and more persistent sclerotia in wet soil (S_wD), and sclerotia density for air‐dried soil (S_dD) were monitored. A logistic model (multiple sigmoid) of disease incidence revealed the lowest parameter values in treatments irrigated daily. Daily frequency of irrigation showed significantly lower disease incidence (39.2%) and disease intensity index (43.9%) and MpD (88.0%) values as areas compared with other frequencies, regardless of the RWC. High RWC significantly reduced (70.8–84.9%) ID, S_wD and S_dD as areas, but significantly increased (18.0%) the incidence of infected plants (IIP), regardless of the irrigation frequency. The disease incidence was not correlated with temperature. Daily irrigation to low RWC mitigated the VWO and the IIP, kept soil to the lowest MpD and resulted in the lowest S_dD level at the end of the trial. Results suggested that less persistent propagules could have played a part in the disease development.     

Phytophthora oleae sp. nov. causing fruit rot of olive in southern Italy

A homothallic Phytophthora species was found to be consistently associated with a rot of mature fruits of two local cultivars of olive (Olea europaea) in Calabria, southern Italy. The phylogenetic analysis of sequences of the ITS1‐5.8S‐ITS2 region and cox1 gene enabled its identification as a new species of clade 2, with a basal position compared to previously described subclades. The new species is described formally with the epithet Phytophthora oleae, referring to the natural matrix from which it was isolated. A unique combination of molecular and morphological characters clearly separates P. oleae from other already described Phytophthora species. This new species produced semipapillate, occasionally bipapillate, persistent sporangia on simple sympodially branching sporangiophores as well as globose and smooth‐walled oogonia, paragynous antheridia and spherical, plerotic oospores. The pathogenicity of P. oleae was confirmed in inoculation trials on fruits of three olive cultivars, including the two local cultivars from which the pathogen had been isolated.     

Variation of pathotypes and races and their correlations with clonal lineages in Verticillium dahliae

Understanding pathogenic variation in plant pathogen populations is key for the development and use of host resistance for managing verticillium wilt diseases. A highly virulent defoliating (D) pathotype in Verticillium dahliae has previously been shown to occur only in one clonal lineage (lineage 1A). By contrast, no clear association has yet been shown for race 1 with clonal lineages. Race 1 carries the effector gene Ave1 and is avirulent on hosts that carry resistance gene Ve1 or its homologues. The hypothesis tested was that race 1 arose once in a single clonal lineage, which might be expected if V. dahliae acquired Ave1 by horizontal gene transfer from plants, as hypothesized previously. In a diverse sample of 195 V. dahliae isolates from nine clonal lineages, all race 1 isolates were present only in lineage 2A. Conversely, all lineage 2A isolates displayed the race 1 phenotype. Moreover, 900‐bp nucleotide sequences from Ave1 were identical among 27 lineage 2A isolates and identical to sequences from other V. dahliae race 1 isolates in GenBank. The finding of race 1 in a single clonal lineage, with identical Ave1 sequences, is consistent with the hypothesis that race 1 arose once in V. dahliae. Molecular markers and virulence assays also confirmed the well‐established finding that the D pathotype is found only in lineage 1A. Pathogenicity assays indicated that cotton and olive isolates of the D pathotype (lineage 1A) were highly virulent on cotton and olive, but had low virulence on tomato.     

Interactions between Trichoderma harzianum and defoliating Verticillium dahliae in resistant and susceptible wild olive clones

Verticillium wilt (VW) in olive is best managed by an integrated disease management strategy, of which use of host resistance is a key element. The widespread occurrence of a highly virulent defoliating (D) Verticillium dahliae pathotype has jeopardized the use of commercial olive cultivars lacking sufficient resistance to this pathogen. However, the combined use of resistant wild olive rootstocks and Trichoderma spp. effective in the biocontrol of VW can improve the management of VW in olive. In vivo interactions between D V. dahliae and Trichoderma harzianum were studied in olive and wild olive plants displaying different degrees of resistance against this pathogen using confocal microscopy. This multitrophic system included wild olive clones Ac‐4 and Ac‐15, olive cv. Picual, and the fungal fluorescent transformants T. harzianum GFP22 and V. dahliae V138I‐YFP, the latter being obtained in this study. In planta observations indicated that V138I‐YFP colonizes the roots and stems of the olive and wild olive genotypes, and that GFP22 grows endophytically within the roots of them all. YFP fluorescence signal quantifications showed that: (i) the degree of root and stem colonization by the pathogen varied depending upon the susceptibility of the tested wild olive genotype, being higher in Ac‐15 than in Ac‐4 plants; and (ii) treatment with T. harzianum GFP22 reduced the extent of pathogen growth in both clones. Moreover, root colonization by strain GFP22 reduced the percentage of pathogen colonies recovered from stems of olive and wild olive plants.     

Phenotypic,molecular and pathogenic characterization of Phlyctema vagabunda,causal agent of olive leprosy

Olive leprosy, caused by the fungus Phlyctema vagabunda, is a classic fruit rot disease widespread in the Mediterranean basin. From 2009 to 2013, new disease symptoms consisting of small circular necrotic leaf lesions, coin branch canker and shoot dieback were observed in Spanish and Portuguese olive orchards showing intense defoliation. Phlyctema‐like anamorphs were consistently isolated from leaves and shoots with symptoms. Representative isolates from affected leaves, shoots and fruits were characterized based on morphology of colonies and conidia, optimum growth temperature and comparison of DNA sequence data from four regions: ITS, tub2, MIT and rpb2. In addition, pathogenicity tests were performed on apple and olive fruits, and on branches and leaves of olive trees. Maximum mycelial growth rate ranged between 0.54 and 0.73 mm per day. Conidia produced on inoculated apple fruits showed slight differences in morphology among the representative fungal isolates evaluated. Phylogenetic analysis clustered all of the Phlyctema‐like isolates in the same clade, identifying them as Phlyctema vagabunda. On fruits, influence of wounding, ripening and cultivar resistance was studied, with cv. Blanqueta being the most susceptible cultivar. On branches, a mycelial‐plug inoculation method reproduced olive leprosy symptoms and caused shoot dieback. On leaves, Koch's postulates were fulfilled and the pathogen caused characteristic necrotic spots and plant defoliation. This is the first time that the pathogenicity of P. vagabunda in olive leaves has been demonstrated.     

Population genetic analysis of Phytophthora infestans in northwestern China

Late blight caused by Phytophthora infestans is the most devastating disease of potato worldwide. To understand the P. infestans population structure and dynamics in northwestern China, 959 single‐lesion isolates were purified in three consecutive years (2009–2011) and were characterized for mating type, pathotype, mtDNA haplotype and molecular variation at eight SSR loci. The results showed that the distribution of mating types changed significantly over years, with self‐fertile isolates dominant in 2010 and 2011. SSR genotyping distinguished 959 isolates into 151 genotypes, and association analysis indicated that P. infestans populations in 2010 and 2011 were strictly asexual while in 2009 they showed signs of sexual reproduction. Population analysis showed that the majority of genetic variation was within P. infestans populations. Isolates sharing identical SSR genotypes were detected in distant regions, indicating that migration of P. infestans could have occurred between regions. Pathogenicity assays on a set of potato differential lines containing R1 to R11 resistance genes detected four pathotypes from 74 selected isolates, with the pathotype virulent against all 11 R genes being dominant. Three mtDNA haplotypes (Ia, IIa, IIb) were detected with Ia being dominant among 507 isolates examined. Phylogenetic analysis indicated that P. infestans populations in northwestern China are distant from European lineages including 13‐A2 (blue‐13) at the time of this survey. The results have implications for the trade of healthy seed tubers as a means of managing late blight.     

Field models for the prediction of leaf infection and latent period of Fusicladium oleagineum on olive based on rain,temperature and relative humidity

Although much is known about the effect of climatic conditions on the development of peacock leaf spot of olive, field‐operational models predicting disease outbreaks are lacking. With the aim of developing such models, a 10‐year survey was conducted to relate leaf infection to climate parameters that can be easily monitored in the field. As outbreaks of disease are known to be linked to rain, models were evaluated for their ability to predict whether infection would occur following a rain event, depending on air temperature and duration of relative humidity above 85%. A total of 134 rain events followed by confirmed leaf infection and 191 rain events not followed by detectable infection were examined. The field data were adequately fitted (both specificity and sensitivity >0·97) with either a multilayer neural network or with two of six tested regression models describing high boundary values of high humidity duration, above which no infection occurred over the temperature range, and low boundary values below which no infection occurred. The data also allowed the selection of a model successfully relating the duration of latent period (time between infection and the first detection of leaf spots) as a function of air temperature after the beginning of rain (R² > 0·98). The predictive abilities of these models were confirmed during 2 years of testing in commercial olive orchards in southern France. They should thus provide useful forecasting tools for the rational application of treatments and foster a reduction in fungicide use against this major disease of olive.     

Phytophthora pachypleura sp. nov., a new species causing root rot of Aucuba japonica and other ornamentals in the United Kingdom

Isolates of an unknown Phytophthora species from the ‘Phytophthora citricola complex’ have been found associated with mortality of Aucuba japonica in the UK. Based on morphological characteristics, growth–temperature relationships, sequences of five DNA regions and pathogenicity assays, the proposed novel species is described as Phytophthora pachypleura. Being homothallic with paragynous antheridia and semipapillate sporangia, P. pachypleura resembles other species in the ‘P. citricola complex’ but can be discriminated by its distinctively thick‐walled oospores with an oospore wall index of 0·71. In the phylogenetic analysis based on three nuclear (ITS, β‐tubulin, EF‐1α) and two mitochondrial (cox1, nadh1) DNA regions, P. pachypleura formed a distinct clade within the ‘P. citricola complex’ with P. citricola s. str., P. citricola E and P. acerina as its closest relatives. Phytophthora pachypleura is more aggressive to A. japonica than P. plurivora and P. multivora and has the potential to affect other ornamental species.     

Aetiology of anthracnose on grapevine shoots in Brazil

Anthracnose is an important disease in vineyards in south and southeast Brazil, the main grape‐producing regions in the country. This study aimed to identify the causal agents of grapevine anthracnose in Brazil through multilocus phylogenetic analyses, morphological characterization and pathogenicity tests. Thirty‐nine Elsinoë ampelina and 13 Colletotrichum spp. isolates were obtained from leaves, stems and berries with anthracnose symptoms collected in 38 vineyards in southern and southeastern Brazil. For E. ampelina isolates, the internal transcribed spacer (ITS), histone H3 (HIS3) and elongation factor 1‐α (TEF) sequences were analysed. HIS3 was the most informative region with 55 polymorphic sites including deletions and substitutions of bases, enabling the grouping of isolates into five haplotypes. Colonies of E. ampelina showed slow growth, variable colouration and a wrinkled texture on potato dextrose agar. Conidia were cylindrical to oblong with rounded ends, hyaline, aseptate, (3.57–) 5.64 (?6.95) μm long and (2.03–) 2.65 (?3.40) μm wide. Seven species of Colletotrichum were identified: C. siamense, C. gloeosporioides, C. fructicola, C. viniferum, C. nymphaeae, C. truncatum and C. cliviae, with a wide variation in colony and conidium morphology. Only E. ampelina caused anthracnose symptoms on leaves, tendrils and stems of Vitis vinifera and V. labrusca. High disease severity and a negative correlation between disease severity and shoot dry weight were observed only when relative humidity was above 95%. In this study, only E. ampelina caused anthracnose symptoms on grapevine shoots in Brazil.     

Molecular and biological characterization of Cowpea mild mottle virus isolates infecting soybean in Brazil and evidence of recombination

The biological and molecular characterization of six isolates of a new Cowpea mild mottle virus strain (CPMMV; Carlavirus, Betaflexiviridae) are reported. Soybean plants with mosaic and stem necrosis were collected in Bahia, Goiás, Mato Grosso and Minas Gerais states, Brazil. Complete genomes of the CPMMV isolates are 8180–8198 nucleotides (nt) long, excluding the 3′‐polyadenylated tail, and have 67–68% nt sequence identity with a Ghana isolate of CPMMV, the only CPMMV isolate for which the genome has previously been sequenced. The replicase has only 60–61% nt sequence identity with the Ghana CPMMV isolate, and the coat protein (CP) is highly conserved (79% nt sequence identity and 95–96% amino acid sequence identity). The high CP identity and the phylogenetic analyses supported the classification of the Brazilian isolates as CPMMV. Biological and molecular differences with the Ghana CPMMV isolate were found and indicated that the six isolates represent a distinct CPMMV strain denominated as CPMMV‐BR. Furthermore, it is shown that recombination occurred mainly in the polymerase gene, and may occur less frequently in other regions of the CPMMV genome.