Danilo B. Pinho

Improving ITS sequence data for identification of plant pathogenic fungi

SummaryPlant pathogenic fungi are a large and diverse assemblage of eukaryotes with substantial impacts on natural ecosystems and human endeavours. These taxa often have complex and poorly understood life cycles, lack observable, discriminatory morphological characters, and may not be amenable to in vitro culturing. As a result, species identification is frequently difficult. Molecular (DNA sequence) data have emerged as crucial information for the taxonomic identification of plant pathogenic fungi, with the nuclear ribosomal internal transcribed spacer (ITS) region being the most popular marker. However, international nucleotide sequence databases are accumulating numerous sequences of compromised or low-resolution taxonomic annotations and substandard technical quality, making their use in the molecular identification of plant pathogenic fungi problematic. Here we report on a concerted effort to identify high-quality reference sequences for various plant pathogenic fungi and to re-annotate incorrectly or insufficiently annotated public ITS sequences from these fungal lineages. A third objective was to enrich the sequences with geographical and ecological metadata. The results – a total of 31,954 changes – are incorporated in and made available through the UNITE database for molecular identification of fungi (http://unite.ut.ee), including standalone FASTA files of sequence data for local BLAST searches, use in the next-generation sequencing analysis platforms QIIME and mothur, and related applications. The present initiative is just a beginning to cover the wide spectrum of plant pathogenic fungi, and we invite all researchers with pertinent expertise to join the annotation effort.

Phylogeny, identification and pathogenicity of the Botryosphaeriaceae associated with collar and root rot of the biofuel plant Jatropha curcas in Brazil, with a description of new species of Lasiodiplodia.

The global expansion of cultivation areas of Jatropha has contributed to the emergence of various diseases. Currently in Brazil, the occurrence of a new disease has been reported that not only reduces the productivity but also causes the death of Jatropha. This disease is associated with collar and root rot of plants. From morphological and phylogenetic studies (based on Internal Transcribed Spacers, β-tubulin and Translation Elongation Factor 1-α sequences), nine species of Botryosphaeriaceae were identified. These species include Lasiodiplodia egyptiacae, L. pseudotheobromae, L. theobromae, Macrophomina phaseolina, Neoscytalidium hyalinum and four Lasiodiplodia spp. that are proposed as new species (L. euphorbicola, L. jatrophicola, L.macrospora and L. subglobosa). All the species in this study, except M. phaseolina, are pathogenic. The results show that root rot of physic nut plants is caused by complex pathogens. This study provides new information for future studies of disease management, quarantine programs and, especially, the development of resistant varieties for collar and root rot disease in J. curcas.

New occurrences of Botryosphaeriaceae causing black root rot of cassava in Brazil

Despite the occurrence of several diseases of cassava, the cassava black root rot (CBR) represents one of the main limiting factor for crop rentability in the world. However, the etiology of CBR is complex and it needs to be revised based on current molecular analysis. On this work, molecular and morphological studies allowed for the identification of three species of Botryosphaeriaceae causing black root rot disease of cassava in the states of Maranhao and Paraiba, Brazil, namely: Lasiodiplodia euphorbicola, Lasiodiplodia pseudotheobromae and Neoscytalidium hyalinum. This is the first report of these three fungal species as causal agents of CBR in the world.

New Meliolaceae from the Brazilian Atlantic forest 1. Species on hosts in the families Asteraceae, Burseraceae, Euphorbiaceae, Fabaceae and Sapindaceae

Five new species, two new varieties and three newly reported taxa belonging to the Meliolaceae were collected in fragments of Atlantic forest from Minas Gerais, Brazil, in association with native plants and are described and illustrated herein. The newly described species are Appendiculella eupatorii, Meliola cassiae-ferrugineae, M. mutisiae, M. peruiferae, M. vernaliae. The new varieties are M. garugae var. protii and M. paullinifolii var. rubiginosae. These taxa are reported in Brazil for the first time: Asteridiella cyclopoda, A. entebbeensis var. codiaei and Meliola pazschkeana var. macropoda. We studied other species belonging in Meliolaceae collected on hosts belonging to the Asteraceae, Burseraceae, Euphorbiaceae, Fabaceae (Caesalpinioideae and Papilionoideae) and Sapindaceae in Brazil.

New Meliolaceae from the Brazilian Atlantic Forest 2: species on host families Annonaceae, Cecropiaceae, Meliaceae, Piperaceae, Rubiaceae, Rutaceae and Tiliaceae

Continuing the study of black mildews in fragments of the Atlantic forest, three new species and five new records are described herein. Irenopsis luheae-grandiflorae, Meliola vicosensis and Meliola xylopia-sericiae are new species. Cecropia hololeuca, Piper gaudichaudianum and Trichilia lepidota are new hosts for Asteridiella leucosykeae, Asteridiella glabroides and Meliola trichiliae respectively. Asteridiella obesa and Meliola psychotriae var. chiococcae are reported for the first time from Brazil. The new species are described and illustrated based on light and scanning electron microscopy and tables with main characteristics of morphologically similar specimens with species collected in Viçosa are provided. Other species belonging to Meliolaceae collected on hosts belonging to the Annonaceae, Meliaceae and Tiliaceae in Brazil also were studied.

First report of Gilbertella persicaria as the cause of soft rot of fruit of Syzygium cumini

A zygomycetous fungus causing fruit soft rot was found on Sygyzium cumini in Northeast Brazil. Based on morphological and phylogenetic analyses, the fungus was identified as Gilbertella persicaria. This is the first report of this fungus causing the decay of S. cumini fruit worldwide.

Naming Potentially Endangered Parasites: Foliicolous Mycobiota of Dimorphandra wilsonii, a Highly Threatened Brazilian Tree Species

A survey of foliicolous fungi associated with Dimorphandra wilsonii and Dimorphandra mollis (Fabaceae) was conducted in the state of Minas Gerais, Brazil. Dimorphandra wilsonii is a tree species native to the Brazilian Cerrado that is listed as critically endangered. Fungi strictly depending on this plant species may be on the verge of co-extinction. Here, results of the pioneering description of this mycobiota are provided to contribute to the neglected field of microfungi conservation. The mycobiota of D. mollis, which is a common species with a broad geographical distribution that co-occurs with D. wilsonii, was examined simultaneously to exclude fungal species occurring on both species from further consideration for conservation because microfungi associated with D. wilsonii should not be regarded as under threat of co-extinction. Fourteen ascomycete fungal species were collected, identified, described and illustrated namely: Byssogene wilsoniae sp. nov., Geastrumia polystigmatis, Janetia dimorphandra-mollis sp. nov., Janetia wilsoniae sp. nov., Johansonia chapadiensis, Microcalliopsis dipterygis, Phillipsiella atra, Piricauda paraguayensis, Pseudocercospora dimorphandrae sp. nov., Pseudocercosporella dimorphandrae sp. nov., Ramichloridiopsis wilsoniae sp. and gen. nov., Stomiopeltis suttoniae, Trichomatomyces byrsonimae and Vesiculohyphomyces cerradensis. Three fungi were exclusively found on D. wilsonii and were regarded as potentially threatened of extinction: B. wilsoniae, J. wilsoniae and R. wilsoniae.

Reappraisal of the black mildews (Meliolales) on Hevea brasiliensis

The rubber tree (Hevea brasiliensis) is host to several fungal species, including Irenopsis heveae as described by Hansford in 1961, which causes black mildew on leaves. One specimen of Irenopsis heveae from the state of Espirito Santo and two from the state of Para were analyzed and showed some morphological differences. Some structures are similar to morphological characteristics as described and illustrated by Vincens in 1915 for Meliola heveae. Morphological comparisons with the type specimen of I. heveae and the alignment of the nucleotide sequences of the 28S rDNA region, however, indicate that the three samples belong to the same species. According to these data M. heveae and I. heveae are heterotypic synonyms with M. heveae being the older name. As the name I. heveae is already occupied by Hansford, Irenopsis vincensii is proposed as new name for the black mildew on H. brasiliensis. This is the first contribution of molecular sequence data for this species.

Black mildew disease on the neotropical medicinal shrub Pothomorphe umbellata in Brazil, caused by Irenopsis tortuosa var. potomorphes

A black mildew disease caused by Irenopsis tortuosa var. potomorphes (Meliolales) was observed on leaves of the neotropical medicinal shrub Pothomorphe umbellata collected in a stretch of Atlantic rain forest in the municipality of Viçosa, Minas Gerais, Brazil. This is the fourth record of Irenopsis tortuosa var. potomorphes on Pothomorphe umbellata and the first record of this fungus in Brazil.

Notes on Ceratocystis paradoxa causing internal post-harvest rot disease on immature coconut in Brazil

Currently there is a large and increasing demand for immature coconuts for fresh consumption of their liquid endosperm (coconut water). The occurrence of post-harvest diseases prevent its sale and change the taste, making it inappropriate for consumption. In 2011, coconut fruits showing internal post-harvest rot symptoms were found in a market in Belo Horizonte and after incubation for 5 days in a humid chamber at room temperature, fungal structures were observed. Fruiting structures show morphology typical of Ceratocystis paradoxa. For molecular identification, DNA sequences were generated for the Internal Transcribed Spacer regions 1 and 2 including the 5.8S rRNA gene, part of the β-tubulin and the Transcription Elongation Factor 1-α gene regions. These data were compared with those of other C. paradoxa using phylogenetic analysis. Kochs postulates was confirmed by inoculation of 6-mm-diameter PDA plugs with the isolate on fruits of coconut. Morphology of the isolates in culture as well as phylogenetic inference showed that the causal agent of internal post-harvest rot disease on coconut is C. paradoxa.