Jadson Diogo Pereira Bezerra

Richness of endophytic fungi isolated from Opuntia ficus-indica Mill. (Cactaceae) and preliminary screening for enzyme production

Opuntia ficus-indica Mill. (forage cactus) is farmed with relative success in the semi-arid region of the Brazilian northeast for commercial purposes, particularly as forage and food. Endophytic microorganisms are those that can be isolated inside plant tissues and can be a new source to production of enzymes with different potentialities. The objective of this study was to describe the richness of endophytic fungi from O. ficus-indica and to detect the capacity of these species to produce extracellular hydrolytic enzymes. Forty-four endophytic fungi species were isolated. Among them, the most commonly found were Cladosporium cladosporioides (20.43%) and C. sphaerospermum (15.99%). Acremonium terricola,Monodictys castaneae, Penicillium glandicola, Phoma tropica and Tetraploa aristata are being reported for the first time as endophytic fungi for Brazil. The majority of isolated fungi exhibited enzymatic potential. Aspergillus japonicus and P. glandicola presented pectinolytic activity. Xylaria sp. was the most important among the other 14 species with positive cellulase activity. All 24 isolates analysed were xylanase-positive. Protease was best produced by isolate PF103. The results indicate that there is a significant richness of endophytic fungi in O. ficus-indica, and that these isolates indicate promising potential for deployment in biotechnological processes involving production of pectinases, cellulases, xylanases and proteases.

Endophytic fungi from medicinal plant Bauhinia forficata: Diversity and biotechnological potential

Bauhinia forficata is native to South America and used with relative success in the folk medicine in Brazil. The diversity, antibacterial activity, and extracellular hydrolytic enzymes of endophytic fungi associated with this plant were studied. Plant samples, which included leaves, sepals, stems, and seeds, were used. Ninety-five endophytic fungal were isolated (18 from leaves, 22 from sepals, 46 from stems, and nine from seeds), comprising 28 species. The most frequently isolated species were Acremonium curvulum (9.5%), Aspergillus ochraceus (7.37%), Gibberella fujikuroi (10.53%), Myrothecium verrucaria (10.53%) and Trichoderma piluliferum (7.37%). Diversity and species richness were higher in stem tissues, and Sorensen’s index of similarity between the tissues was low. Eleven fungi showed antibacterial activity. Aspergillus ochraceus , Gibberella baccata , Penicillium commune , and P. glabrum were those with the greatest antibacterial activity against Staphylococcus aureus and/or Streptococcus pyogenes . Thirteen species showed proteolytic activity, particularly Phoma putaminum . Fourteen species were cellulase positive, particularly the Penicillium species and Myrmecridium schulzeri . All isolates tested were xylanase positive and 10 showed lipolytic activity, especially Penicillium glabrum . It is clear that the endophytic fungi from B. forficata have potential for the production of bioactive compounds and may be a source of new therapeutic agents for the effective treatment of diseases in humans, other animals, and plants. To our knowledge, this is the first study of endophytic fungi from different tissues of B. forficata and their biotechnological potential.

Bezerromycetales and Wiesneriomycetales ord. nov. (class Dothideomycetes), with two novel genera to accommodate endophytic fungi from Brazilian cactus

During a survey of endophytic fungi from the cactus Tacinga inamoena in a Brazilian tropical dry forest (Caatinga) some undescribed ascomycetous fungi were isolated. These fungi are characterized by superficial and immersed, globose to subglobose, smooth or hairy ascomata, bitunicate asci, and muriformly septate, ellipsoidal ascospores. Multigene phylogenetic analyses using sequences from partial ITS, SSU and LSU nrDNA and the translation elongation factor 1-alpha gene (tef1) demonstrated a monophyletic clade accommodating these endophytic fungi in the class Dothideomycetes, closely related to the order Tubeufiales. Based on morphological features and phylogenetic analyses, these fungi could not be placed in the order Tubeufiales, in the new order Wiesneriomycetales, or any other known genus in the class Dothideomycetes. Thus, two new genera (Bezerromyces, with B. brasiliensis and B. pernambucoensis, and Xiliomyces with X. brasiliensis), a new family (Bezerromycetaceae) and a new order (Bezerromycetales) are introduced to accommodate these novel taxa. Our phylogenetic analyses also demonstrated that the clade accommodating Wiesneriomycetaceae represents a new order, here introduced as Wiesneriomycetales.

Phylogeny and taxonomy of the scab and spot anthracnose fungus Elsinoë (Myriangiales, Dothideomycetes)

Species of Elsinoë are phytopathogens causing scab and spot anthracnose on many plants, including some economically important crops such as avocado, citrus, grapevines, and ornamentals such as poinsettias, field crops and woody hosts. Disease symptoms are often easily recognisable, and referred to as signature-bearing diseases, for the cork-like appearance of older infected tissues with scab-like appearance. In some Elsinoë-host associations the resulting symptoms are better described as spot anthracnose. Additionally the infected plants may also show mild to severe distortions of infected organs. Isolation of Elsinoë in pure culture can be very challenging and examination of specimens collected in the field is often frustrating because of the lack of fertile structures. Current criteria for species recognition and host specificity in Elsinoë are unclear due to overlapping morphological characteristics, and the lack of molecular and pathogenicity data. In the present study we revised the taxonomy of Elsinoë based on DNA sequence and morphological data derived from 119 isolates, representing 67 host genera from 17 countries, including 64 ex-type cultures. Combined analyses of ITS, LSU, rpb2 and TEF1-α DNA sequence data were used to reconstruct the backbone phylogeny of the genus Elsinoë. Based on the single nomenclature for fungi, 26 new combinations are proposed in Elsinoë for species that were originally described in Sphaceloma. A total of 13 species are epitypified with notes on their taxonomy and phylogeny. A further eight new species are introduced, leading to a total of 75 Elsinoë species supported by molecular data in the present study. For the most part species of Elsinoë appear to be host specific, although the majority of the species treated are known only from a few isolates, and further collections and pathogenicity studies will be required to reconfirm this conclusion.

New endophytic Toxicocladosporium species from cacti in Brazil, and description of Neocladosporium gen. nov.

Brazil harbours a unique ecosystem, the Caatinga, which belongs to the tropical dry forest biome. This region has an important diversity of organisms, and recently several new fungal species have been described from different hosts and substrates within it. During a survey of fungal endophyte diversity from cacti in this forest, we isolated cladosporium-like fungi that were subjected to morphological and multigene phylogenetic analyses including actA, ITS, LSU, rpb2 and tub2 gene sequences. Based on these analyses we identified two new species belonging to the genus Toxicocladosporium, described here as T. cacti and T. immaculatum spp. nov., isolated from Pilosocereus gounellei subsp. gounellei and Melocactus zehntneri, respectively. To improve the species recognition and assess species diversity in Toxicocladosporium we studied all ex-type strains of the genus, for which actA, rpb2 and tub2 barcodes were also generated. After phylogenetic reconstruction using five loci, we differentiated 13 species in the genus. Toxicocladosporium velox and T. chlamydosporum are synonymized based on their phylogenetic position and limited number of unique nucleotide differences. Six strains previously assigned to T. leucadendri, including the ex-type strain (CBS 131317) of that species, were found to belong to an undescribed genus here named as Neocladosporium gen. nov., with N. leucadendri comb. nov. as type species. Furthermore, this study proposes the actA, ITS, rpb2 and tub2 as main phylogenetic loci to recognise Toxicocladosporium species.

Pathogenicity of Beauveria bassiana and production of cuticle-degrading enzymes in the presence of Diatraea saccharalis cuticle

The sugarcane borer, Diatraea saccharalis , is one of the worst pests in Brazilian sugarcane crop, causing high levels of financial losses every year. Beauveria bassiana is an entomopathogenic fungus widely used in the biological control of several agricultural pests. The aims of this study were to: (1) evaluate the pathogenicity of B. bassiana strains against D. saccharalis (2) investigate the production of proteases and chitinase by B. bassiana in the presence of the cuticle of sugarcane borer; and, (3) evaluate the relation between the production of enzymes and pathogenicity of the strains. All isolates tested were pathogenic to D. saccharalis and the mortality ranged from 36 to 88%. The production of enzymes was higher in the medium containing cuticle, showing that the process is stimulated by specific components found in the cuticle of the host. Pr1 activity was higher than Pr2 and both were produced at 24 h. The highest production of chitinase was obtained at 96 h of culture for all strains tested. Levels of specific cuticle-degrading enzymes such as proteases correlated positively with specific virulence parameters. B. bassiana URM2915 showed promising features to be used in a biological control program of D. saccharalis . Key words : Biological control, sugarcane, subtilisin-like protease, trypsin-like protease, chitinase.

Families and genera of diaporthalean fungi associated with canker and dieback of tree hosts

In this study we accept 25 families in Diaporthales based on phylogenetic analyses using partial ITS, LSU, rpb2 and tef1-α gene sequences. Four different families associated with canker and dieback of tree hosts are morphologically treated and phylogenetically compared. These include three new families (Diaporthostomataceae, Pseudomelanconidaceae, Synnemasporellaceae), and one new genus, Dendrostoma (Erythrogloeaceae). Dendrostoma is newly described from Malus spectabilis, Osmanthus fragrans and Quercus acutissima having fusoid to cylindrical, bicellular ascospores, with three new species namely D. mali, D. osmanthi and D. quercinum. Diaporthostomataceae is characterised by conical and discrete perithecia with bicellular, fusoid ascospores on branches of Machilus leptophylla. Pseudomelanconidaceae is defined by conidiogenous cells with apical collarets and discreet annellations, and the inconspicuous hyaline conidial sheath when mature on Carya cathayensis, compared to morphologically similar families Melanconidaceae and Juglanconidaceae. Synnemasporellaceae is proposed to accommodate fungi with synnematous conidiomata, with descriptions of S. toxicodendri on Toxicodendron sylvestre and S. aculeans on Rhus copallina.

Diaporthe from walnut tree ( Juglans regia ) in China, with insight of the Diaporthe eres complex

Species of Diaporthe are important plant pathogenic fungi that commonly occur on a wide range of hosts. They are relatively difficult to identify due to their extreme similarity in morphology and confusing multigene phylogeny, especially in the Diaporthe eres complex. In the present study, isolates were collected from diseased branches of Juglans regia in China. Most strains were clustered into the D. eres species complex based on the combined internal transcribed spacer (ITS) region, partial calmodulin (CAL), histone H3 (HIS), translation elongation factor 1-alpha (TEF1-α) and beta-tubulin (TUB) genes. To focus on this complex, CAL, TEF1-α and TUB were selected in further phylogenetic analyses that showed a better topology compared with combined five-gene phylogeny. Results revealed that all strains which clustered in the Diaporthe eres complex from Juglans regia in China were Diaporthe eres. Results suggested a revised species criterion in the Diaporthe eres complex. The current study uncovered a new species here described as Diaporthe. tibetensis.

Mycological Diversity Description I

Here, Quambalaria fabacearum and Neopestalotiopsis brasiliensis are introduced as new species from Brazil, isolated as endophyte from Mimosa tenuiflora and causing post-harvest rot disease on fruits of Psidium guajava, respectively. Diaporthe inconspicua is emended to include a more detailed morphological description. Neopestalotiopsis egyptiaca is reported as new to the Americas and as causing post-harvest rot disease on fruits of Psidium guajava, while Umbelopsis isabellina is reported as endophyte.

First report of Gilbertella persicaria causing soft rot in eggplant fruit in Brazil

In March 2017, two eggplant fruit (Solanum melongena cv. Cica) were collected from field production in Cha-Grande, in the state of Pernambuco, Brazil, with circular, light brown and slightly sunken lesions in the apex, extending to the entire fruit in 3 to 4 days and developing a soft rot with abundant mycelial and sporangia production on the surface. After microscopic examination, a fungus belonging to the genus Gilbertella was identified. The fungus was isolated from soft rot observed in the fruit and pure cultures were obtained by transferring a single spore to potato dextrose agar (PDA). Two isolates were obtained and deposited in the culture collection “Micoteca URM Profa. Maria Auxiliadora Cavalcanti” at the Universidade Federal de Pernambuco (Recife, Brazil), as URM7670 and URM7671. Morphological characteristics include spherical, single sporangia with 57.2 to 96.2 µm diameter, yellow-brown to brown when young turning dark brown or black on maturity, and sporangia split longitudinally into two halves to release the spore and expose a columella obvoid, 23.4 to 65 × 26 to 52 µm. The sporangiospores were mostly globose to ellipsoid, smooth, hyaline, aseptate, and 7.8 to 13 × 5.2 to 10.4 µm. The morphological characteristics evaluated corroborate previously published data for Gilbertella persicaria (Benny 1991). The partial sequences of the internal transcribed spacer (ITS) and 28S (LSU) of the rDNA were obtained for the two isolates and deposited in GenBank (accession nos. MF804516, MF804517, MG573061, and MG573062, respectively). The isolates showed a 99% similarity with ITS and LSU sequences of the type strain G. persicaria CBS 190.32 (NR111692 and JN939197, respectively) according to BLAST search. A combined phylogenetic tree obtained by Bayesian method grouped the isolates with the G. persicaria clade with 100% of posterior probability, confirming the identification. Koch’s postulates were conducted by inoculating five asymptomatic eggplant fruit (cv. Cica) previously disinfested with 0.5% hypochlorite and unwounded or superficially wounded with a sterile needle at two equidistant points. Ten milliliters of a sporangiospore suspension (1 × 10⁵ sporangiospores/ml) obtained from a representative isolate (URM7670) were sprayed on the inoculation sites. Sterile distilled water was used as control. The inoculated fruits were maintained in plastic boxes that contained a portion of moistened cotton wool and were maintained in a moist chamber at ∼25°C for 5 days. Wounded inoculated fruit showed the first symptoms (circular, light brown spots) 48 h after inoculation and evolved into soft rot after 5 days. Hyaline hyphae and light brown to dark sporangia were observed over the surface of the infected fruit. The pathogen was reisolated, fulfilling Koch’s postulates. The control and unwounded fruit remained asymptomatic. This species is usually found in soils and dung, but has already been reported to cause rot in peach fruit (Ginting et al. 1996), dragon fruit (Guo et al. 2012), black plum (Pinho et al. 2014), and papaya (Cruz-Lachica et al. 2016). To our knowledge, G. persicaria represents a new report in the world associated with soft rot in eggplant. This work will certainly contribute to the knowledge of pathogens that affect eggplant culture and, consequently, future studies that involve disease management strategies.