Identification of potential hosts plants of Cowpea aphid‐borne mosaic virus

Among the major pathogens affecting passion fruit orchards, the cowpea aphid‐borne mosaic virus (CABMV), also known as the fruit‐hardening virus, has gained prominence owing to its role in the drastic reduction in fruit production in yellow passion fruit orchards (Passiflora edulis f. flavicarpa) from the second year of cultivation. To mitigate the damage, several regions adopt the annual planting system where a sanitary void is maintained from August to September. However, the virus is believed to remain dormant in weeds. This study aimed to identify potential weed hosts of CABMV. The study was conducted with a randomized design with four replications in Londrina, PR. Twenty‐eight weed species were tested, and a sample of yellow passion fruit leaves symptomatic for CABMV infection was used as the virus inoculum source. Mechanical inoculation was performed using the extract from the symptomatic plant. Symptoms were visually evaluated every 3 days. For molecular confirmation, total RNA was extracted, followed by RT‐PCR with CABMV‐specific oligonucleotides, reinoculation in passion fruit plants and sequencing. CABMV infection was observed in southern sandbur (Cenchrus echinatus), Siberian motherwort (Leonurus sibiricus), showy rattlepod (Crotalaria spectabilis) and yellow passion fruit (Passiflora edulis). The CABMV‐positive weed species extract was able to infect yellow passion fruit plant when a fresh mechanical inoculation was performed. Showy rattlepod (Crotalaria spectabilis) was the only weed species to exhibit observable symptoms of CABMV. C. echinatus, L. sibiricus and C. spectabilis act as a source of CABMV inoculum.     

Rust disease of eucalypts,caused by Puccinia psidii,did not originate via host jump from guava in Brazil

The rust fungus, Puccinia psidii, is a devastating pathogen of introduced eucalypts (Eucalyptus spp.) in Brazil where it was first observed in 1912. This pathogen is hypothesized to be endemic to South and Central America and to have first infected eucalypts via a host jump from native guava (Psidium guajava). Ten microsatellite markers were used to genotype 148 P. psidii samples from eucalypts and guava plus five additional myrtaceous hosts across a wide geographic range of south‐eastern Brazil and Uruguay. Principal coordinates analysis, a Bayesian clustering analysis and a minimum‐spanning network revealed two major genetic clusters among the sampled isolates, one associated with guava and another associated with eucalypts and three additional hosts. Multilocus genotypes infecting guava differed by multiple mutational steps at eight loci compared with those infecting eucalypts. Approximate Bayesian computation revealed that evolutionary scenarios involving a coalescence event between guava‐ and eucalypt‐associated pathogen populations within the past 1000 years are highly unlikely. None of the analyses supported the hypothesis that eucalypt‐infecting P. psidii in Brazil originated via host jump from guava following the introduction of eucalypts to Brazil approximately 185 years ago. The existence of host‐associated biotypes of P. psidii in Brazil indicates that this diversity must be considered when assessing the invasive threat posed by this pathogen to myrtaceous hosts worldwide.     

Host Specificity and Genetic Diversity of Didymella bryoniae from Cucurbitaceae in Brazil

Thirty‐seven isolates of Didymella bryoniae from three Cucurbitaceae species were collected in Brazil and tested for pathogenicity to watermelon. All isolates were pathogenic but differed in aggressiveness levels. Seven representative isolates were used in cross‐pathogenicity tests against 10 cucurbitaceous hosts. Most isolates were pathogenic to most host species tested, except to Sechium edule. Among the susceptible species, Citrullus and Cucumis species were the most susceptible hosts, while pumpkin and Luffa purgans were the most resistant. Host of origin affected the pattern of aggressiveness on each host. Isolates from watermelon were very aggressive to their original host, but much less aggressive or not pathogenic at all to some Cucurbita. Two previously described random‐amplified polymorphic DNA (RAPD)‐specific primers indicated that 81% of the isolates could be classified into the so‐called RG I group, while the remaining isolates could not be classified into any of the described RG groups. All 37 isolates were further characterized by RAPD fingerprinting and compared with three US isolates representative of RG I and RG II groups. The Brazilian D. bryoniae isolates could be separated into genetically similar clusters. The majority of the isolates were grouped in cluster DB Ia, which contained only isolates of Citrullus lanatus and Cucumis melo. Two of the American isolates used as controls clustered with this group at 68% similarity level. The DB Ib cluster included three Brazilian isolates obtained from melon and watermelon and the American representative for RG II, at a lower similarity level (43%). Two isolates from watermelon clustered with one isolate from melon in a separate group (DB II), while one single isolate from pumpkin (DB III) showed the lowest genetic similarity to all other isolates. Didymella bryoniae isolates from Brazil showed, therefore, a level of genetic diversity higher than previously reported for the species. RAPD fingerprinting allowed for geographical distinction of D. bryoniae isolates but no correlation between genetic distance, aggressiveness or origin of the isolate was found.     

Rapid detection of cowpea aphid-borne mosaic virus in cowpea seeds

Four cultivars of cowpea (Vigna unguiculata [L]. Walp.) were infected with cowpea aphid-borne mosaic virus (CABMV) by natural infection in field plots. Seeds taken from these plants were tested for the presence of the virus by ELISA and symptom observation on the plantlets grown from the seeds. A biotin/ streptavidin ELISA technique was used and found to be more sensitive than a standard ELISA protocol for detecting CABMV infection in seed. There was a good correlation between the ELISA detection of CABMV in tissue taken from single cowpea seeds and subsequent development of infected plants grown from the same seeds. The ELISA technique is reliable for selecting CABMV-free stocks of cowpea seeds.     

Inheritance of resistance to potyviruses in Phaseolus vulgaris L. IV. Inheritance, linkage relations, and environmental effects on systemic resistance to four potyviruses

We have examined the genetics of systemic resistance in Phaseolus vulgaris to azuki bean mosaic virus (AzMV) and cowpea aphid-borne mosaic virus (CABMV) and the relationship of this resistance to a phenotypically similar resistance to watermelon mosaic virus (WMV) and soybean mosaic virus (SMV). In P. vulgaris cv Great Northern 1140 (GN1140), resistance to SMV and WMV has been attributed to the genes Smv and Wmv, respectively, which have been shown to segregate as a unit. Systemic resistance to AzMV is conferred by two incompletely dominant alleles, Azm1 and Azm2, at unlinked loci. At least three resistance alleles must be present at these two loci for systemic resistance to be expressed in the plant. Systemic resistance to CABMV in GN 1140 is conditioned by a dominant allele that has been designated Cam2. Under some environmental conditions, a recessive allele at an unlinked locus, cam3, also controls a resistant response to CABMV. Resistance to AzMV and CABMV does not assort independently from Wmv/Smv, but also does not consistently cosegregate, suggesting that perhaps in each case one of the factors involved in resistance is associated with Smv/Wmv.     

Phylogenetic Analysis of Uromyces Species Infecting Grain and Forage Legumes by Sequence analysis of Nuclear Ribosomal Internal Transcribed Spacer Region

DNA sequence analysis of the nuclear ribosomal internal transcribed spacer region (ITS) was performed to determine phylogenetic relationship between 49 isolates of rusts infecting grain and forage legumes. Isolates were collected from different hosts and distinct geographic origins and represent eight species of Uromyces: U. anthyllidis, U. appendiculatus, U. ciceris‐arietini, U. minor, U. pisi, U. striatus, U. viciae‐fabae and U. vignae. ITS sequences revealed length polymorphisms and variation in DNA sequence that were used to characterize phylogenetic relationships by maximum parsimony, maximum likelihood and Bayesian analyses which in general agreed revealing the presence of four clearly distinct clades. Clade one included the isolates causing rust on chickpea, fenugreek and alfalfa. Clade two was composed by rust isolates of field clover and pea plants, while the third clade was formed by bean and cowpea isolates. Clade four was the largest and included all the rust isolates infecting faba bean. Within this clade, the highly supported subclusters of U. viciae‐fabae collected on Lens culinaris, U. viciae‐fabae collected on Vicia sativa and U. viciae‐fabae collected on Lathyrus palustris suggest an ongoing process of host specialization.     

Responses of different geographic populations of two potato tuber moth species to genetic variants of Phthorimaea operculella granulovirus

Phthorimaea operculella granulovirus (PhopGV) belongs to the genus Betabaculovirus of the arthropod‐infecting Baculoviridae. PhopGV is able to infect several gelechiid species. Among them are the potato tuber moths Phthorimaea operculella Zeller and Tecia solanivora Povolny (both Lepidoptera: Gelechiidae). In various South American countries, PhopGV‐based biopesticides are used to control either P. operculella or T. solanivora. Many trials have indicated that a particular viral isolate can exhibit very distinct pathogenicity when infecting different host species or different populations of one host species. In this study, we compared host–pathogen interactions using various PhopGV isolates and various populations of P. operculella and T. solanivora. Virus isolates from P. operculella were more pathogenic against their original host species than against T. solanivora. A PhopGV isolated from T. solanivora was less efficient against P. operculella. In addition, virus isolates differed in pathogenicity toward their hosts (i.e., lethal concentrations of isolates ranged from low to high). Unexpectedly, we also found that host populations of one species from distinct geographic origins did not differ significantly in susceptibility to the same PhopGV isolate. This was the case for both host species and for five PhopGV isolates. Comparative restriction fragment length polymorphism (RFLP) analyses of 11 isolates including those used in bio‐assays indicated three main regions of variation in the genome of PhopGV, corresponding to the regions of open reading frame PhopGV046, gene PhopGV129 (egt), and repeat 9 (located between open reading frames PhopGV083 and PhopGV084). Comparison of the nucleotide sequences of the insertions/deletions present in these regions were carried out for the most variable isolate, JLZ9f. The results are discussed in the context of the production and use of PhopGV as a biological agent against these two pest species.     

Identification and Characterization of a Potyvirus Isolated from Siratro Plants

The present work describes the identification and characterization of a potyvirus isolated from siratro (Macroptilium atropurpureum Urb.) in the north‐west region of the State of São Paulo, Brazil. The virus was transmitted by mechanical inoculation. Its host range was restricted mainly to members of the Fabaceae. A cDNA fragment of about 930 bp was amplified by RT/PCR, cloned and sequenced. The fragment, which included the coat protein gene, had amino acid identity percentages between 88 and 98% with isolates of Bean common mosaic virus (BCMV). Phylogenetic analysis grouped the siratro potyvirus and BCMV isolates in 99% of the replicates, including Azuki mosaic virus, Dendrobium mosaic virus, Blackeye cowpea mosaic virus and Peanut stripe virus, which have been classified as BCMV strains. This is the first citation on the presence of BCMV in siratro plants in Brazil.     

Alarming increase in the incidence of Cucumber mosaic virus in cowpea (Vigna unguiculata (L.) Walp.) in northern Nigeria

Cowpea plays a key nutritional role in the diet of the Nigerian people. Viral diseases are a major limitation to cowpea production worldwide, and thus, constant viral surveillance is crucial for monitoring and management purposes. In this study, cowpea leaf samples from fields in three northern Nigeria states, Kano, Kaduna and Niger, were tested to determine the status of six common viruses previously reported in these cowpea-producing states following the release of virus-resistant varieties. Cowpea aphid-borne mosaic virus (CABMV), Blackeye cowpea mosaic virus (BICMV), Cowpea mottle virus, Southern bean mosaic virus and Cucumber mosaic virus (CMV) were detected. Cowpea yellow mosaic virus, which was previously reported in all three states, was not detected in any of the samples tested, while CMV that was previously regarded as unimportant to cowpea production in Nigeria had the highest incidence in all three states, and the overall highest incidence of 58.8%, while CABMV had the lowest incidence (7.5%). CMV was also present in seven of the ten mixed infection combinations detected. Dual infection of CMV and BICMV, which often results in cowpea stunt, the most devastating cowpea disease in the USA, was the most frequently detected mixed infection (28.1%) and was detected in all three states. This observed elevation in CMV infection in cowpea must be closely monitored and swiftly managed to avert possible devastating crop yield losses.     

Identification and Characterization of Colletotrichum spp. affecting Fruit after Harvest in Brazil

Colletotrichum spp. cause anthracnose in various fruits post‐harvest and are a particularly important problem in tropical and subtropical fruits. The disease in fruits of avocado, guava, papaya, mango and passion fruit has been reported to be caused by C. gloeosporioides, and in banana by C. musae. In subtropical and temperate crops such apple, grape, peach and kiwi, the disease is caused by C. acutatum. The variation in pathogenic, morphological, cultural and molecular characteristics of Brazilian isolates of Colletotrichum acutatum Simmonds and isolates from post‐harvest decays of avocado, banana, guava, papaya, mango and passion fruit was evaluated. The fruits were inoculated with mycelium of C. acutatum, Colletotrichum spp. and C. musae on a disc of potato dextrose agar. The morphological, cultural and molecular characteristics studied were conidia morphology, colony growth at different temperatures, colony coloration and PCR with primers CaInt2 and ITS4 for C. acutatum and CgInt and ITS4 for C. gloeosporioides. C. acutatum was pathogenic to avocado, guava, papaya, mango and passion fruit, but it was not pathogenic to banana. The morphological, cultural and molecular studies indicated that the avocado, papaya, mango and passion fruit isolates were C. gloeosporioides. The natural guava isolate was identified as C. acutatum, which had not been found previously to produce anthracnose symptoms on guava in Brazil.     

Sequence Diversity of the Nucleoprotein Gene of Peanut bud necrosis virus Isolates from South India

Peanut bud necrosis virus (PBNV), genus Tospovirus (family Bunyaviridae), is an important virus infecting peanut and other crops in South India. PBNV isolates naturally infecting groundnut, brinjal, tomato, black gram, field bean, cowpea, cotton, jute, taro and Calotropis plants were collected from different regions of South India and characterized. Infection was confirmed by direct antigen‐coating enzyme‐linked immunosorbent assay (DAC‐ELISA) using PBNV‐specific antiserum. The coat protein gene was further amplified using PBNV coat protein‐specific primers. The amplicon (830 bp) was cloned and sequenced; sequence analysis revealed that the N gene shared 93–100% and 95–100% sequence identity with PBNV at the nucleotide and amino acid levels, respectively.     

Host range and genetic strains of leafminer flies (Diptera: Agromyzidae) in eastern Brazil reveal a new divergent clade of Liriomyza sativae

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Amphibian‐killing chytrid in Brazil comprises both locally endemic and globally expanding populations

Chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis (Bd), is the emerging infectious disease implicated in recent population declines and extinctions of amphibian species worldwide. Bd strains from regions of disease‐associated amphibian decline to date have all belonged to a single, hypervirulent clonal genotype (Bd‐GPL). However, earlier studies in the Atlantic Forest of southeastern Brazil detected a novel, putatively enzootic lineage (Bd‐Brazil), and indicated hybridization between Bd‐GPL and Bd‐Brazil. Here, we characterize the spatial distribution and population history of these sympatric lineages in the Brazilian Atlantic Forest. To investigate the genetic structure of Bd in this region, we collected and genotyped Bd strains along a 2400‐km transect of the Atlantic Forest. Bd‐Brazil genotypes were restricted to a narrow geographic range in the southern Atlantic Forest, while Bd‐GPL strains were widespread and largely geographically unstructured. Bd population genetics in this region support the hypothesis that the recently discovered Brazilian lineage is enzootic in the Atlantic Forest of Brazil and that Bd‐GPL is a more recently expanded invasive. We collected additional hybrid isolates that demonstrate the recurrence of hybridization between panzootic and enzootic lineages, thereby confirming the existence of a hybrid zone in the Serra da Graciosa mountain range of Paraná State. Our field observations suggest that Bd‐GPL may be more infective towards native Brazilian amphibians, and potentially more effective at dispersing across a fragmented landscape. We also provide further evidence of pathogen translocations mediated by the Brazilian ranaculture industry with implications for regulations and policies on global amphibian trade.     

Molecular evidence for host-adapted races of the fungal endophyte Epichloë bromicola after presumed host shifts

Abstract.— Host shifts of plant‐feeding insects and parasites promote adaptational changes that may result in the formation of host races, an assumed intermediate stage in sympatric speciation. Here, we report on genetically differentiated and host‐adapted races of the fungal endophyte Epichloë bromicola, which presumably emerged after a shift from the grass Bromus erectus to other Bromus hosts. Fungi of the genus Epichloë (Ascomycota) and related anamorphs of Neotyphodium are widespread endophytes of cool‐season grasses. Sexually reproducing strains sterilize the host by formation of external fruiting structures (stromata), whereas asexual strains are asymptomatic and transmitted via seeds. In E. bromicola, strains infecting B. erectus are sexual, and strains from two woodland species, B. benekenii and B. ramosus, are asexual and seed transmitted. Analyses of amplified fragment length polymorphism fingerprinting and of intron sequences of the tub2 and tef1 genes of 26 isolates from the three Bromus hosts collected at natural sites in Switzerland and nearby France demonstrated that isolates are genetically differentiated according to their host, indicating that E. bromicola does not form a single, randomly mating population. Phylogenetic analyses of sequence data did not unambiguously resolve the exact origin of asexual E. bromicola strains, but it is likely they arose from within sexual populations on B. erectus. Incongruence of trees derived from different genes may have resulted from recombination at some time in the recent history of host strains. Reciprocal inoculations of host plant seedlings showed that asexual isolates from B. benekenii and B. ramosus were incapable of infecting B. erectus, whereas the sexual isolates from B. erectus retained the assumed ancestral trait of broad compatibility with Bromus host seedlings. Because all isolates were interfertile in experimental crosses, asexual strains may not be considered independent biological species. We suggest that isolates infecting B. benekenii and B. ramosus represent long‐standing host races or incipient species that emerged after host shifts and that may evolve through host‐mediated reproductive isolation toward independent species.     

Morphological and genetic characterization of bloom‐forming Raphidophyceae from Brazilian coast

Massive fish kills caused by bloom‐forming species of the Raphidophyceae occur in many marine coastal areas and often cause significant economic losses. The ultrastructure and phylogeny of marine raphidophytes from the Brazilian coast have not been fully analyzed. Here, we present the first combined morphological and genetic characterization of raphidophyte strains from the Brazilian coast. Ten strains of four raphidophyte species (Chattonella subsalsa, C. antiqua, Heterosigma akashiwo, and Fibrocapsa japonica) were characterized based on morphology (including ultrastructure) and LSU rDNA sequences. Chattonella subsalsa and C. antiqua formed two distinct genetic clades. We found that the cell size is the only phenotypic feature separating C. subsalsa and C. antiqua strains from Brazil, whereas traditional characteristics used for species separation in the genus Chattonella (i.e., tail size, chloroplast presence in the tail, ‘oboe‐shaped’ mucocysts, and presence of thylakoids in the pyrenoid matrix) were not sufficiently discriminative, due to their overlapping in the two taxa. The phylogenetic analysis indicated intra‐specific geographic differences among C. subsalsa sequences, with two subclades: one formed by isolates from Brazil, USA, and Iran, and another by a sequence from the Adriatic Sea (Italy). Fibrocapsa japonica also showed intra‐specific geographic differences, with a sequence from a Brazilian strain grouped with strains from Japan, Australia, and Germany, all of them distinct from the Italian isolates. This is the first combined morphological and phylogenetic analysis of raphidophytes from the South Atlantic. Our findings broaden knowledge of the biodiversity of this important bloom‐forming algal group.     

EXPERIMENTAL EVOLUTION OF AN EMERGING PLANT VIRUS IN HOST GENOTYPES THAT DIFFER IN THEIR SUSCEPTIBILITY TO INFECTION

This study evaluates the extent to which genetic differences among host individuals from the same species condition the evolution of a plant RNA virus. We performed a threefold replicated evolution experiment in which Tobacco etch potyvirus isolate At17b (TEV‐At17b), adapted to Arabidopsis thaliana ecotype Ler‐0, was serially passaged in five genetically heterogeneous ecotypes of A. thaliana. After 15 passages we found that evolved viruses improved their fitness, showed higher infectivity and stronger virulence in their local host ecotypes. The genome of evolved lineages was sequenced and putative adaptive mutations identified. Host‐driven convergent mutations have been identified. Evidences supported selection for increased translational efficiency. Next, we sought for the specificity of virus adaptation by infecting all five ecotypes with all 15 evolved virus populations. We found that some ecotypes were more permissive to infection than others, and that some evolved virus isolates were more specialist/generalist than others. The bipartite network linking ecotypes with evolved viruses was significantly nested but not modular, suggesting that hard‐to‐infect ecotypes were infected by generalist viruses whereas easy‐to‐infect ecotypes were infected by all viruses, as predicted by a gene‐for‐gene model of infection.     

Root and Crown Rot of Brazilian Pine (Araucaria angustifolia) Caused by Phytophthora cinnamomi

In an area reforested with Brazilian pine (Araucaria angustifolia) located in Paraná State, southern Brazil, 20‐ to 40‐year‐old trees representing 0.2% of the surveyed area had symptoms of root and crown rot, yellowing and browning of leaves from the uppermost branches and death. Three Phytophthora isolates obtained from diseased plant tissue were tested against 1‐year‐old Brazilian pine seedlings and found to display positive pathogenicity. Based on their morphological and physiological characteristics, the isolates were identified as Phytophthora cinnamomi. A GenBank BLAST search of partial sequences from the β‐tubulin and elongation factor‐1α genes, as well as the ITS regions and 5.8S gene of rDNA, confirmed the species identification. This is the first report of the involvement of this pathogen on the aetiology of Brazilian pine root and crown rot.     

Dolichos yellow mosaic virus belongs to a distinct lineage of Old World begomoviruses; its biological and molecular properties

Dolichos yellow mosaic disease (DYMD) affects the production of dolichos in South Asia. Diseased plants produce characteristic bright yellow mosaic patches on the leaves and early infections cause reductions in yield. The putative dolichos yellow mosaic virus (DoYMV) was transmitted poorly (maximum 18.3% transmission) by the whitefly, Bemisia tabaci. DoYMV has a narrow host range and infected only Lablab purpureus and L. purpureus var. typicum out of the 36 species tested. Virus was detected using monoclonal antibodies in a triple‐antibody sandwich enzyme‐linked immunosorbent assay and by PCR. Complete DNA‐A components of DoYMV isolates from Mysore and Bangalore, South India, were sequenced, but several attempts to identify DNA‐B and DNA‐β were unsuccessful. DoYMV isolates shared DNA‐A nucleotide identities of 92.5–95.3% with previously described isolates from North India and Bangladesh. They were most similar to mungbean‐infecting begomoviruses at 61.6–64.4% of DNA‐A nucleotide identities. Phylogenetic analyses of DNA‐A sequences grouped the dolichos‐infecting and mungbean‐infecting begomoviruses into a distinct cluster away from begomoviruses infecting non‐leguminous plants in the Indian subcontinent. Antigenically, legume‐infecting begomoviruses were most similar to each other compared with non‐legume viruses. Collectively, these results indicate that legume‐infecting begomoviruses in the Indian subcontinent belonged to a distinct lineage of Old World begomoviruses.     

The true identity of Obama (Platyhelminthes: Geoplanidae) flatworm spreading across Europe

Since 2008 there have been many records in Europe (British Isles, Spain, France, Italy) of a large terrestrial planarian morphologically very similar to the Brazilian species Obama marmorata. Sequences of mitochondrial (Cox1) and nuclear (18S, 28S, ITS‐1 and EF) genes from European specimens and some from Brazil indicate that they belong to a species different from that of other specimens also collected in Brazil. Moreover, the phylogenetic results show that they are not sister‐species. Histological sections of both Brazilian and European specimens reveal subtle morphological differences between the two species. Obama marmorata is confined to Brazil, and the second, herein described new species, O bama nungara sp. nov. , is found in Brazil and Europe. These cryptic species may be syntopic in areas in Brazil. The new species occurs in human‐modified environments both in Brazil and in Europe. We also conclude that the specimens from Spain and Argentina identified previously as Obama marmorata belong to the new species.