Heterogeneity of Pseudomonas savastanoi populations infecting Myrtus communis in Sardinia (Italy)

Genetic, phenotypic and host range diversity among Pseudomonas savastanoi isolates from Myrtus communis were investigated. Thirty‐one isolates from six Sardinian commercial myrtle orchards and three isolates from plants growing spontaneously on the island of Rhodes (Greece) were compared with reference strains of P. savastanoi from olive, oleander, ash and myrtle. Multilocus sequence analysis (MLSA) indicated the presence of a monomorphic population with a very low level of variability. Conversely, Biolog phenotypic fingerprinting and phytohormone production analyses showed a considerable metabolic diversity, as bacteria obtained from single infected tissue differed more than bacteria obtained from different orchards. When pathogenicity tests were carried out on myrtle plants, different types of symptoms were induced: knots, canker lesions with or without tissue proliferations and, occasionally, wilting of the inoculated twig, a symptom never reported before for P. savastanoi. Comparable symptoms were also observed in the natural environment both on spontaneous and cultivated plants. Moreover, the host range of the myrtle population was heterogeneous and not well defined. Some isolates showed a wide host range whilst others were pathogenic only to their natural host. Overall these findings suggest that the diversity of the P. savastanoi population from myrtle does not depend so much on the locality or the natural host and does not allow the Sardinian and Greek isolates, together with previously characterized myrtle strains, to be ascribed to a known pathovar of P. savastanoi, nor to propose their belonging, as a whole, to a new pathovar.     

The colonization processes of Myrtus communis by strains of Pseudomonas savastanoi with a differential ability to produce phytohormones

The ability to produce indole-3-acetic acid (IAA) through the indole-3-acetamide (IAM) pathway as well as cytokinins is a common trait of Pseudomonas savastanoi populations causing disease on oleander and olive. These phytohormones are required for the induction and development of an outgrowth of plant cell tissue termed a knot. However, in myrtle orchards of Sardinia (Italy), strains of P. savastanoi unable to produce cytokinins were found coexisting with cytokinin-producing strains. Data presented here show that the ability to produce IAA through the IAM pathway is also a variable trait within this population, raising questions on the exact role of these plant growth substances in the disease process on myrtle. Three P. savastanoi strains were selected based on their differential ability to produce phytohormones in vitro, and their interaction with the host was investigated over a period of 8 months using histological methods. All strains successfully invaded the infected twigs, moving systemically (unhalted by host defences) upward and downward from the inoculation point, both by completely degrading the cell walls and by taking advantage of the xylem vessels and intercellular spaces. Moreover, all strains induced the development of cankers, which slowly evolved into typical knots only on the twigs inoculated with the phytohormone-producing strains. This study further demonstrates that cytokinins and IAA are essential for knot development; moreover, it ascertains that bacterial production of cytokinins is not necessary for host colonization and for the expression of pathogenicity (i.e. the ability to cause disease) of P. savastanoi on myrtle.     

Monitoring the occurrence of tomato bacterial spot and range of the causal agent Xanthomonas perforans in Iran

A 2‐year comprehensive field survey was conducted across major tomato‐growing areas of Iran. Two hundred and thirty‐four tomato fields and six tomato‐producing greenhouses were surveyed for the potential presence of bacterial spot disease. Five hundred and ninety‐six tomato samples with and without symptoms were analysed. While Xanthomonas spp. were found in association with tomato plants both with and without symptoms from five surveyed counties, the bacterial spot disease was observed only in plants from three of them. Only strains isolated from plants with symptoms induced disease symptoms on tomato, while those isolated from symptomless plants caused symptoms only on cabbage and common bean. None of the isolates caused disease symptoms on pepper and eggplant. Phylogenetic analysis showed that X. perforans is the causal agent of tomato bacterial spot in Iran, although X. campestris and X. axonopodis were also associated with symptomless tomato plants. All X. perforans isolates in this study were sensitive to streptomycin, copper sulphate and copper oxychloride at concentrations of 50 mg L^?1, 200 mg L^?1 and 0.8 g L^?1, respectively. Unlike the type strain of X. perforans, isolates in this study did not produce bacteriocin against other Xanthomonas spp., nor were they detected using the usual species‐specific primer pair Bs‐XpF/Bs‐XpR. This suggests an atypical nature of X. perforans strains in Iran, which leads to the hypothesis that X. perforans strains in Iran may have a separate origin to those causing disease epidemics elsewhere. The aggregated dispersal pattern of the diseased tomato fields signifies the seedborne introduction of the pathogen into the country.     

Dissemination of Pseudomonas savastanoi pv. savastanoi populations and subsequent appearance of olive knot disease

Pseudomonas savastanoi pv. savastanoi (Psv) is the causal agent of olive knot disease. The bacterium survives epiphytically and gains ingress through new wounds where infections and colonization result in knot formation. The natural spread of the bacterium and the subsequent appearance of the disease in olive orchards is poorly understood. The aim of this study was to monitor Psv epiphytic populations in inoculated plants with knots versus non‐inoculated healthy trees within the same orchard over four years. Additionally, disease severity was measured in both inoculated and non‐inoculated control trees. Epiphytic Psv populations moved from inoculated to non‐inoculated trees, although average Psv populations were higher in inoculated trees. Olive knot severity increased over the course of the study in all treatments and cultivars, with all plants reaching a high level of disease by the end of the study. However, the delay in the onset of disease was longer in non‐inoculated than in inoculated trees. Molecular typing of Psv isolates recovered from non‐inoculated control trees confirmed that they were similar to the inoculated strain. These data demonstrate that Psv can move over short distances in olive orchards through dissemination of epiphytic bacteria and suggest a relationship between the presence of epiphytic Psv and the number of knots on trees.     

Population structure,genetic diversity,and sexual state of the rice brown spot pathogen Bipolaris oryzae from three Asian countries

Bipolaris oryzae causes brown spot in rice (Oryza sativa) inflicting substantial grain yield losses worldwide. Knowledge of the population structure, genetic diversity and sexual recombination of the fungal pathogen can help to implement effective disease management strategies. In this study, B. oryzae isolates sampled from Iran, the Philippines and Japan were analysed with 12 simple‐sequence repeat (SSR) markers, newly developed from the genome sequence of the fungus. Among the 288 B. oryzae isolates genotyped, 278 unique haplotypes were identified. High genotype numbers (richness) with even distribution (evenness) were found within the collection sites. Both mating types, MAT1‐1 and MAT1‐2, were present in each collection area, and the sexual state was induced under controlled conditions with production of viable ascospores. However, the tests of linkage disequilibrium rejected of the hypothesis of random mating. Discriminant analysis of principal components (DAPC) revealed that the B. oryzae collection formed three clusters, each consisting of isolates from different collection sites. Analysis of molecular variance (amova ) showed that genetic variation among clusters was 18.7%, with the rest of the variation distributed within clusters (R_ST = 0.187, P < 0.001). Statistically significant pairwise genetic differentiation was found between the clusters. These results show that Asian B. oryzae isolates are genetically diverse, and, overall, distributed in three groups. These findings will be helpful in managing the disease and guide the use of representative isolates needed for selection of resistant rice varieties.     

Pathogenic and molecular comparison of Puccinia kuehnii isolates and reactions of sugarcane varieties to orange rust

Sugarcane orange rust, a disease caused by Puccinia kuehnii, was first reported in Brazil in 2009. There are no studies comparing the Brazilian P. kuehnii collections and the reaction of important sugarcane varieties under controlled conditions. This work compared the reaction of seven sugarcane varieties inoculated with six different P. kuehnii isolates from Brazilian sugarcane areas and verified the pathogenic and genetic variability of these isolates. The incubation (I) and latency (L) disease periods, disease severity (SEV), total number of lesions (TNL), total number of sporulating lesions (TNSL), and percentage of sporulating lesions (%SL) were evaluated. Furthermore, ITS1 and IGS ribosomal sequences of all P. kuehnii isolates used in this study were compared with pathogen sequences from 13 different countries. The disease incubation ranged from 7 to 10 days and the latency ranged from 10 to 21 days. SEV and TNL showed large variations and few significant differences between the reaction of the varieties to P. kuehnii, in contrast with the variables TNSL and %SL. The P. kuehnii isolates did not compose different virulent races, but the isolate from one site (Araras) was a more aggressive race. The ITS1 and IGS ribosomal sequences of six P. kuehnii isolates were identical with each other and to most P. kuehnii American sequences deposited at GenBank. The studied sequences of P. kuehnii isolates differed from the sequences from Asia, Tahiti and Oceania.     

Pathotypic and genetic diversity in the population of Rhizoctonia solani AG1‐IA causing rice sheath blight in China

Sheath blight, caused by Rhizoctonia solani AG1‐IA, is one of the most serious diseases of rice. In this study, a total of 175 isolates of R. solani AG1‐IA were collected from five rice‐growing regions in China. Pathogenicity tests revealed that all isolates were virulent to five cultivars with different levels of resistance at the rice seedling stage in the greenhouse. There was considerable variation in aggressiveness, and the isolates were classified into three pathotypes based on disease severity, with moderately virulent isolates prevalent in the population. Forty‐three haplotypes were identified based on ITS sequencing, and 39 haplotypes were distinct among isolates. There were high levels of haplotype diversity and nucleotide diversity within the populations of R. solani AG1‐IA. High gene flow (Nm = 1·63–5·22) was detected, consistent with relatively low differentiation between pairs of populations. Five populations were divided into two distinct clusters by the unweighted pair group method with arithmetic mean (UPGMA), and no spatial population differentiation was discernible. The majority (97·8%) of genetic diversity was distributed among isolates within populations, with only 2·2% of the genetic diversity attributed to differences among populations. The star‐like shape of the haplotype network provided evidence of signatures of population expansion in recent history. No significant relationships were found between the genetic diversity and aggressiveness or geographic origin among populations of R. solani AG1‐IA. These results highlight that the population characteristics of R. solani AG1‐IA should be taken into account in evaluating the germplasm resistance of rice cultivars to sheath blight.     

Large subclonal variation in Phytophthora infestans populations associated with Ecuadorian potato landraces

The population of Phytophthora infestans on potato landraces in three provinces (Carchi, Chimborazo and Loja) of Ecuador was analysed. All isolates (n = 66) were of the A1 mating type. Simple sequence repeats (SSR) were used to assess the genetic diversity of the isolates. The P. infestans isolates from the potato landraces grouped in a single clade together with reference isolates belonging to the clonal lineage EC‐1. In the 66 SSR profiles obtained, 31 multilocus genotypes were identified. The 66 isolates constituted 49 different races according to the Solanum demissum differential set ( R1 to R11). The P. infestans population was complex and virulent on 4 to 11 R genes. Analysis showed that the subclonal variation in the Ecuadorian EC‐1 clone is increasing over time and is much larger than clonal variation in lineages in the Netherlands and Nicaragua, suggesting high mutation rates and little or no selection in Ecuador.     

Non‐pathogenic rhizosphere bacteria belonging to the genera Rhizorhapis and Sphingobium provide specific control of lettuce corky root disease caused by species of the same bacterial genera

Lettuce corky root (CR) is caused by bacteria in the genera Rhizorhapis, Sphingobium, Sphingopyxis and Rhizorhabdus of the family Sphingomonadaceae. Members of this family are common rhizosphere bacteria, some pathogenic to lettuce. Sixty‐eight non‐pathogenic isolates of bacteria obtained from lettuce roots were tested for control of CR caused by Rhizorhapis suberifaciens CA1^T and FL1, and Sphingobium mellinum WI4^T. In two initial screenings, 10 isolates significantly reduced CR induced by one or more pathogenic strains on lettuce seedlings in vermiculite, while seven non‐pathogenic isolates provided significant CR control in natural or sterilized field soil. Rhizorhapis suberifaciens FL11 was effective at controlling all pathogenic strains, but most effective against R. suberifaciens CA1^T. The other selected isolates controlled only pathogenic strains belonging to their own genus. In a greenhouse experiment, a soil drench with selected biocontrol agents (R. suberifaciens FL11, Sphingomonas sp. NY3 and S. mellinum CA16) controlled CR better than seed treatments or application of alginate pellets. In microplots infested with R. suberifaciens CA1^T, seed treatment with R. suberifaciens FL11 provided complete control and a soil drench with FL11 significantly reduced the disease. Pathogenicity tests with FL11 on 23 plant species in 10 families resulted in slight yellowing on roots of lettuce and close relatives; similar yellowing appeared on some roots of non‐inoculated lettuce plants. This research showed that biocontrol agents can be genus‐specific. Only one isolate, FL11, provided more general control of various pathogenic strains causing CR even in field soil in pots and microplots.     

Pathogenic variation of Mycosphaerella species infecting banana and plantain in Nigeria

Mycosphaerella species that cause the ‘Sigatoka disease complex’ account for significant yield losses in banana and plantain worldwide. Disease surveys were conducted in the humid forest (HF) and derived savanna (DS) agroecological zones from 2004 to 2006 to determine the distribution of the disease and variation among Mycosphaerella species in Nigeria. Disease prevalence and severity were higher in the HF than in the DS zone, but significant (P < 0·001) differences between agroecological zones were only observed for disease severity. A total of 85 isolates of M. fijiensis and 11 isolates of M. eumusae were collected during the survey and used to characterize the pathogenic structure of Mycosphaerella spp. using a putative host differential cultivar set consisting of Calcutta‐4 (resistant), Valery (intermediate) and Agbagba (highly susceptible). Area under disease progress curve (AUDPC) was higher on all cultivars when inoculated with M. eumusae than with M. fijiensis, but significant (P < 0·05) differences between the two species were only observed on Valery. Based on the rank‐sum method, 8·3% of the isolates were classified as highly aggressive and 46·9% were classified as aggressive. About 11·5% of all the isolates were classified as least aggressive, and all of these were M. fijiensis. The majority of M. eumusae isolates (seven out of 11; 64%) were classified as aggressive. A total of nine pathotype clusters were identified using cluster analysis of AUDPC. At least one M. fijiensis isolate was present in all the nine pathotype clusters, while isolates of M. eumusae were present in six of the nine clusters. Isolates in pathotype clusters III and V were the most aggressive, while those in cluster VIII were the least aggressive. Shannon’s index (H) revealed a more diverse Mycosphaerella collection in the DS zone (H = 1·81) than in the HF (H = 1·50) zone, with M. fijiensis being more diverse than M. eumusae. These results describe the current pathotype structure of Mycosphaerella in Nigeria and provide a useful resource that will facilitate screening of newly developed Musa genotypes for resistance against two important leaf spot diseases of banana and plantain.     

Phenotypic and phylogenetic studies associated with the crucifer white leaf spot pathogen,Pseudocercosporella capsellae,in Western Australia

Pseudocercosporella capsellae (white leaf spot disease) is an important disease on crucifers. Fifty‐four single‐conidial isolates collected from Brassica juncea (Indian mustard), B. napus (oilseed rape), B. rapa (turnip), and Raphanus raphanistrum (wild radish) across Western Australia were investigated for differences in pathogenicity and virulence using cotyledon screening tests, genetic differences using internal transcribed spacer (ITS) sequencing and phylogenetic analysis, and growth rates on potato dextrose, V8 juice and malt extract agars. All isolates from the four crucifer hosts were pathogenic on the three test species: B. juncea, B. napus and R. raphanistrum, but showed differences in levels of virulence. Overall, isolates from B. juncea, B. napus and B. rapa showed greatest virulence on B. juncea, least on R. raphanistrum and intermediate virulence on B. napus. Isolates from R. raphanistrum showed greatest virulence on B. juncea, least on B. napus and intermediate virulence on R. raphanistrum. Growth and production of a purple‐pink pigment indicative of cercosporin was greatest on malt extract agar and cercosporin production on V8 juice agar was positively correlated with virulence of isolates on B. juncea and B. napus. ITS sequencing and phylogenetic analysis showed that isolates collected from B. napus, B. juncea and B. rapa, in general and with few exceptions, had a high degree of genetic similarity. In contrast, isolates from R. raphanistrum were clearly differentiated from isolate groups collected from Brassica hosts. Pseudocercosporella capsellae reference isolates from other countries generally grouped into a single separate cluster, highlighting the genetic distinctiveness of Western Australian isolates.     

Inoculation of Malus genotypes with a set of Erwinia amylovora strains indicates a gene‐for‐gene relationship between the effector gene eop1 and both Malus floribunda 821 and Malus ‘Evereste’

The Gram‐negative bacterium Erwinia amylovora, causal agent of fire blight disease in pome fruit trees, encodes a type three secretion system (T3SS) that translocates effector proteins into plant cells that collectively function to suppress host defences and enable pathogenesis. Until now, there has only been limited knowledge about the interaction of effector proteins and host resistance presented in several wild Malus species. This study tested disease responses in several Malus wild species with a set of effector deletion mutant strains and several highly virulent E. amylovora strains, which are assumed to influence the host resistance response of fire blight‐resistant Malus species. The findings confirm earlier studies that deletion of the T3SS abolished virulence of the pathogen. Furthermore, a new gene‐for‐gene relationship was established between the effector protein Eop1 and the fire blight resistant ornamental apple cultivar Evereste and the wild species Malus floribunda 821. The results presented here provide new insights into the host–pathogen interactions between Malus sp. and E. amylovora.     

Genetic diversity of Botrytis in New Zealand vineyards and the significance of its seasonal and regional variation

Species‐ and population‐specific differences in fungicide resistance and aggressiveness within Botrytis makes basic data on genetic diversity important for understanding disease caused by this fungus. Genetic diversity of Botrytis was surveyed between 2008 and 2012 from grapes from five New Zealand wine‐growing regions. A total of 1226 isolates were gathered from symptomless flower buds at the start of the growing season and 1331 isolates from diseased fruit at harvest. Two species were found, B. cinerea and B. pseudocinerea. Botrytis pseudocinerea was common in both Auckland vineyards sampled, and infrequent elsewhere. However, even in Auckland, it was rarely isolated from diseased fruit. The presence of the Boty and Flipper transposons was assessed. Isolates with all four transposon states (Boty only, Flipper only, both Boty and Flipper, no transposons) were found for both species. Both vineyards in the Auckland region had high numbers of Flipper‐only isolates at flowering; both vineyards from the Waipara region had high numbers of Boty‐only isolates at flowering. Most isolates from diseased fruit at harvest contained both transposons. These observations suggest that B. pseudocinerea, and isolates with one or both of the transposons missing, may be less aggressive than B. cinerea, or than isolates with both transposons present. Two clades were resolved within B. pseudocinerea, only one of which has been reported from European vineyards. Phylogenetic diversity within B. cinerea in New Zealand was similar to that known from Europe, including isolates that appear to match Botrytis ‘Group S’. The taxonomic implications of this genetic diversity are discussed.     

Species of the Colletotrichum gloeosporioides and C. boninense complexes associated with olive anthracnose

The taxonomic status of Colletotrichum gloeosporioides sensu lato (s.l.) associated with olive anthracnose is still undetermined and the pathogenic ability of this species complex is controversial. In the present study, isolates obtained from olive and provisionally identified as C. gloeosporioides s.l. on the basis of morphological and cultural features were reclassified using ITS and TUB2 as DNA barcode markers and referred to seven distinct species, recently separated within C. gloeosporioides (C. aenigma, C. gloeosporioides sensu stricto (s.s.), C. kahawae, C. queenslandicum, C. siamense and C. theobromicola) and C. boninense (C. karstii) species complexes. Furthermore, isolates of C. kahawae were ascribed to the subspecies ciggaro by analysing the GS gene. A single isolate, not in either of these two species complexes, was not identified at the species level. In pathogenicity tests on detached olive drupes some of these species, including C. aenigma, C. kahawae subsp. ciggaro, C. queenslandicum, C. siamense and C. karstii, were shown to be weakly pathogenic. Moreover, they were found very sporadically on olive. In contrast, some isolates of C. gloeosporioides s.s. and isolates of C. theobromicola proved to be virulent on both green and ripening olives. This study gives a better insight into both the aetiology and the epidemiology of olive anthracnose and might have implications for biosecurity and quarantine because C. theobromicola has never been reported in major European olive‐producing countries.     

Modification of a multiple‐locus variable number tandem repeat analysis (MLVA) for typing isolates of Erwinia amylovora

Erwinia amylovora, the causal agent of fire blight that affects economically important rosaceous plants, is reported among the most important plant pathogenic bacteria. The low genetic diversity within E. amylovora and the lack of simple and high‐resolution genotyping techniques make epidemiology and evolutionary studies challenging for this pathogen. A multiple‐locus variable number tandem repeat analysis (MLVA) based on a set of nine variable number tandem repeat loci was successfully used to type 46 E. amylovora isolates collected from different host plants in 16 countries, mainly Mediterranean. The nine polymorphic loci proved to have high discriminatory power and to increase the resolution of the MLVA. Thirty‐eight haplotypes clustered in seven clonal complexes. The results identified potentially useful genetic markers among the Mediterranean strains, particularly from the Balkan Peninsula and the Eastern Mediterranean countries. Different MLVA types were observed amongst Italian strains only, indicating the possibility of multiple introductions of the disease. MLVA can be used effectively as a fast, cheap, and simple tool to track E. amylovora infection sources, to gain insight into geographic diversity, and to understand the dynamic evolution of the pathogen.     

Phoma leaf spot of wasabi (Wasabia japonica) caused by Leptosphaeria biglobosa

A leaf spot disease on wasabi plants grown in commercial greenhouses in the Fraser Valley of British Columbia was characterized. Mycelial growth and pycnidial formation were observed within lesions when leaves were incubated under conditions of high humidity. Isolation from diseased tissues consistently yielded colonies of a Phoma species. Sequence analysis of the rDNA internal transcribed spacer (ITS1‐5.8S‐ITS2) region of eight isolates showed 100% nucleotide sequence identity with Phoma wasabiae and Leptosphaeria biglobosa subspecies ‘occiaustralensis’ and 99.2% identity with L. biglobosa ‘canadensis’. Pathogenicity studies on wasabi leaves showed that wounding greatly facilitated infection and enhanced lesion development for most isolates but was not required for all isolates. Chlorotic areas appeared around the inoculation sites within 4 days, followed by necrosis. Isolates displayed a range of virulence, from weakly to highly virulent, on wasabi leaves. Similar results were observed on leaves of canola cultivar Westar, i.e. wounding significantly increased lesion size and isolates displayed a range of virulence. An isolate of Leptosphaeria maculans ‘brassicae’ from canola was highly virulent on wasabi and canola leaves, causing lesions similar to those of L. biglobosa ‘occiaustralensis’ while an isolate of L. biglobosa ‘canadensis’ from canola was weakly virulent on both hosts and required wounds to infect. These results demonstrate that isolates of L. biglobosa ‘occiaustralensis’ from wasabi are as virulent as L. biglobosa ‘canadensis’ on wasabi and canola leaves but in some cases were comparable in virulence to L. maculans ‘brassicae’.     

Characterization and epidemiology of Colletotrichum acutatum sensu lato (C. chrysanthemi) causing Carthamus tinctorius anthracnose

In 2012, Colletotrichum isolates were collected from field‐grown safflower (Carthamus tinctorius) crops in central Italy from plants exhibiting typical anthracnose symptoms. Colletotrichum isolates were also collected from seed surfaces and from within seeds. The genetic variability of these isolates was assessed by a multilocus sequencing approach and compared with those from Colletotrichum chrysanthemi and Colletotrichum carthami isolates from different geographic areas and other Colletotrichum acutatum sensu lato‐related isolates. Phylogenetic analysis revealed that all of the strains isolated from C. tinctorius belonged to the species described as C. chrysanthemi, whereas all of the strains belonging to C. carthami had been isolated from Calendula officinalis. Phenotypic characterization of isolates was performed by assessing growth rates at different temperatures, morphology of colonies on potato dextrose agar (PDA) and the size of conidia. All C. chrysanthemi isolates from safflower had similar growth rates at different temperatures, comparable colony morphologies when grown on PDA and conidial sizes consistent with previously described C. chrysanthemi isolates. Pathogenicity tests were performed by artificially inoculating both seeds and plants and confirmed the seedborne nature of this pathogen. When inoculated on plants, C. chrysanthemi caused the typical symptoms of anthracnose on leaves. This is the first record of this pathogen on C. tinctorius in Italy, and it presents an updated characterization of Colletotrichum isolates pathogenic to safflowers in Europe.     

Biological and molecular variation of Cherry leaf roll virus isolates from Malus domestica,Ribes rubrum,Rubus idaeus,Rumex obtusifolius and Vaccinium darrowii

Cherry leaf roll virus (CLRV) isolates from Malus domestica, Ribes rubrum, Rubus idaeus, Rumex obtusifolius and Vaccinium darrowii were characterized based on nucleotide sequences of a 371 bp fragment of the 3′ untranslated region (UTR) of their genomic RNAs, symptoms in the herbaceous hosts, Chenopodium amaranticolor, Chenopodium quinoa, Nicotiana benthamiana, Nicotiana occidentalis and Nicotiana tabacum, and seed transmission in N. occidentalis. The different isolates induced a range of localized and systemic disease symptoms, of varying severity, in the herbaceous hosts. The isolates from M. domestica, R. rubrum, R. obtusifolius and V. darrowii all showed greater than 80% seed transmission in N. occidentalis, but no seed transmission was observed for the R. idaeus isolate. Based on symptoms and seed transmission, the isolates appear to be biologically distinct strains of CLRV. Phylogenetic analysis of the nucleotide sequences from the 3′ UTR, commonly used to detect CLRV, showed that four isolates from M. domestica, R. rubrum, R. idaeus and V. darrowii were almost identical but an isolate from R. obtusifolius exhibited a pairwise nucleotide difference of up to 5·4% when compared to these isolates. There was no obvious correlation between sequence differences and symptomatology.     

Population structure and migration of the witches’ broom pathogen Moniliophthora perniciosa from cacao and cultivated and wild solanaceous hosts in southeastern Brazil

Moniliophthora perniciosa, causal agent of witches’ broom disease in cacao plantations in South America and the Caribbean Islands, has co‐evolved with its host cacao, but the pathogen has also emerged in many solanaceous hosts in Brazil, including economically important food crops and wild species. This study was carried out to: (i) determine the existence of host subpopulations of M. perniciosa in Brazil; (ii) estimate gene and genotypic diversity of M. perniciosa host subpopulations infecting solanaceous hosts in southeastern Bahia and Minas Gerais states, Brazil; and (iii) estimate the amount and directionality of historical migration of M. perniciosa subpopulations. Up to 203 M. perniciosa isolates collected from solanaceous hosts with symptoms from Bahia and Minas Gerais states in Brazil and from Theobroma spp. (cacao) and Herrania spp. were characterized with 11 microsatellite markers. Factorial correspondence analyses, minimum‐spanning network and Bayesian clustering revealed genetic clusters associated with their host of origin. Significant subpopulation differentiation was evident (Φ_ST = 0.30, P ≤ 0.05) among M. perniciosa host subpopulations. Most of the multilocus microsatellite genotypes (MLMGs) were host‐specific, with few MLMGs shared among subpopulations. Pairwise comparisons among M. perniciosa host subpopulations were significant, except between jurubeba (Solanum paniculatum) and cultivated solanaceous subpopulations. The combined analyses rejected the null hypothesis that M. perniciosa in Brazil is a single genetic population not structured by host. These findings support a scenario of introduction and subsequent adaptation to solanaceous hosts that should be taken into consideration to improve mitigation and management of M. perniciosa.     

Contrasting species boundaries between sections Alternaria and Porri of the genus Alternaria

The fungal genus Alternaria comprises a large number of asexual taxa with diverse ecological, morphological and biological modes ranging from saprophytes to plant pathogens. Understanding the speciation processes affecting asexual fungi is important for estimating biological diversity, which in turn affects plant disease management and quarantine enforcement. This study included 106 isolates of Alternaria representing five phylogenetically defined clades in two sister sub‐generic groups: section Porri (A. dauci, A. solani and A. limicola) and section Alternaria (A. alternata/tenuissima and A. arborescens). Species in section Porri are host‐specific while species in section Alternaria have wider host ranges. For each isolate, DNA sequences of three genes (Alt a1, ATPase, Calmodulin) were used to estimate phylogenies at the population and species levels. Three multilocus haplotypes were distinguished among A. dauci isolates and only one haplotype among A. solani and A. limicola isolates, revealing low or no differentiation within each taxon and strong clonal structure for taxa in this section. In contrast, 37 multilocus haplotypes were found among A. alternata/tenuissima isolates and 21 multilocus haplotypes among A. arborescens isolates, revealing much higher genotypic diversity and multiple clonal lineages within taxa, which is not typical of asexual reproducing lineages. A species tree was inferred using a Yule Speciation model and a strict molecular clock assumption. Species boundaries were well defined within section Porri. However, species boundaries within section Alternaria were only partially resolved with no well‐defined species boundaries, possibly due to incomplete lineage sorting. Significant association with host specificity seems a driving force for speciation.