Trichothecene Genotype Profiling of Wheat Fusarium graminearum Species Complex in Paraguay.

Paraguay is a non-traditional wheat-producing country in one of the warmest regions in South America. Fusarium Head Blight (FHB) is a critical disease affecting this crop, caused by the Fusarium graminearum species complex (FGSC). A variety of these species produce trichothecenes, including deoxynivalenol (DON) and its acetylated forms (3-ADON and 15-ADON) or nivalenol (NIV). This study characterized the phylogenetic relationships, and chemotype diversity of 28 strains within FGSC collected from wheat fields across different country regions. Phylogenetic analysis based on the sequence of elongation factor-1α gene (EF-1α) from 28 strains revealed the presence of four species in the FGSC: F. graminearum sensu stricto, F. asiaticum, F. meridionale and F. cortaderiae. Ten strains selected for further analysis revealed that all F. graminearum strains were 15-ADON chemotype, while the two strains of F. meridionale and one strain of F. asiaticum were NIV chemotype. Thus, the 15-ADON chemotype of F. graminearum sensu stricto was predominant within the Fusarium strains isolated in the country. This work is the first report of phylogenetic relationships and chemotype diversity among Fusarium strains which will help understand the population diversity of this pathogen in Paraguay.

Cryptococcus depauperatus, a close relative of the human-pathogen C. neoformans, associated with coffee leaf rust (Hemileia vastatrix) in Cameroon.

The genus Cryptococcus is well known for its two species -Cryptococcus neoformans and C. gatii- that are etiological agents of cryptococcosis, an important fungal disease of mammals, including humans, and which is particularly common in immunocompromised patients. Nevertheless, Cryptococcus is a large and widely distributed genus of basidiomycetes occupying a broad range of niches, including mycoparasitism. One such mycoparasitic species is Cryptococcus depauperatus, which was firstly mistakenly described as a pathogen of scale insects under the name Aspergillus depauperatus. The "Aspergillus" conidiophores were later shown to be basidia of a Cryptococcus and the new combination C. depauperatus was proposed. Additionally, instead of an entomopathogen, the fungus was found to be a mycoparasite growing on the entomopathogen Akanthomyces (Lecanicillium) lecanii. Recently, during surveys for mycoparasites of coffee leaf rust (Hemileia vastatrix) in the context of a biocontrol project, white colonies covering rust pustules were observed in Cameroon. Upon close examination, instead of a member of the "white colony forming complex" of Ascomycetes, commonly collected growing on H. vastatrix, such colonies were found to represent a basidiomycete fungus with basidia-bearing chains of basidiospores, typical of the genus Cryptococcus. Morphological and molecular evidence was generated supporting the identification of the fungus on rust pustules as C. depauperatus. This is the first record of C. depauperatus from Africa and of its association with coffee leaf rust.

Elucidating the interactions between the rust Hemileia vastatrix and a Calonectria mycoparasite and the coffee plant.

Calonectria hemileiae, a fungus associated with pustules of the coffee leaf rust (CLR, Hemileia vastatrix) in Brazil, was tested in vitro and in planta to assess its biocontrol potential. The fungus inhibited the germination of rust spores by over 80%. CLR severity was reduced by 93% when Calonectria was applied to coffee leaf discs inoculated with H. vastatrix, whilst a reduction of 70-90% was obtained for in planta experiments. Mycoparasitism was demonstrated through the fulfillment of Koch's postulates. Elucidation of the biochemical interaction between Calonectria and Hemileia on coffee plants indicated that the mycoparasite was able to increase plant resistance to rust infection. Coffee plants sprayed with Calonectria alone showed greater levels of chitinase, ß-1,3-glucanase, ascorbate peroxidase and peroxidase. Although effective in controlling the rust, fungicide applications damaged coffee photosynthesis, whereas no harm was caused by Calonectria. We conclude that C. hemileiae shows promise as a biocontrol agent of CLR.

First report of Dematophora bunodes causing root rot of taro (Colocasia esculenta ) and leatherleaf fern (Rumohra adiantiformis) in Brazil.

Colocasia esculenta, taro (T), is a major staple food crop in the tropics, including Brazil. Rumohra adiantiformis, leatherhead fern (LF), is broadly cultivated for its ornamental fronds that are used as a component of flower arrangements. Soft root rot of T and LF, and accompanying rapid plant wilt and death, was observed in plantations in Espírito Santo (Brazil), at Venda Nova do Imigrante, in April 2014 (LF) and July 2015 (T). Great losses were observed. Firstly, a few individual scattered plants showed symptoms of disease in the plantations, then aggregates of plants and, after a few seasons, the majority of the plants in the field died before harvest, leading to the abandonment of the activity by farmers. A white mycelial matt was observed on the crown and roots ofying T and LF plants. Infected corms become necrotic and dark brown mycelial strands were observed internally in tissues. Diseased organs were carefully washed and surface sterilized in 10% sodium hypochlorite. Samples of tissue were removed from the boundary of necrotic tissues and placed on potato dextrose-agar (PDA) plates and incubated at 23±2 C in the dark. Homogeneous mycelial colonies were isolated from both T and LF and, upon observation of microscope mounts under an Olympus BX 53 light microscope, pear-shaped hyphal swellings at the septae (Castro et al. 2013) were observed. . A representative isolate from each host was deposited in the local culture collection as COAD 2911 (LF isolate) and COAD 2912 (T isolate). Additionally, DNA was extracted from each culture using the Wizard Genomic DNA Purification Kit (Promega) and the internal transcriptional spacer region was PCR amplified using the primers ITS5 and ITS1 (White et al. 1990). The amplicons were sequenced by MACROGEN (http://www.macrogen.com). Consensus sequences were deposited in GenBank: MW561595 (LF), MW561596 (T). Consensus regions were compared against other sequences available in Genbank. A BLASTn analysis resulted in LF and T sequences respectively 99% (526/531bp) and 98% (412/420 bp) identity with that of Dematophora bunodes (MN984619). Additionally, a phylogenetic analysis of a selected sequence alignment was performed on the CIPRES webportal (Miller et al., 2010) using MrBayes v.3.1.1 (Ronquist & Huelsenbeck, 2003). A phylogenetic tree was generated showing that the placement of LF and T isolates is in D. bunodes (Wittstein et al. 2020). Pathogenicity tests were performed for LF and T isolates against their original hosts. For inoculum, bags of twice-autoclaved parboiled rice were seeded separately with each isolate, which were allowed to colonize the rice for two weeks. Four healthy young LF and T plants were utilized. Two extra healthy plants grown in the same conditions, but not inoculated, served as controls. Thirty g of Dematophora-colonized rice was placed in direct contact with stems or roots of each LF or T plant. Plants were maintained in a dew chamber for 48 h after inoculation and then transferred to a greenhouse bench. All inoculated plants developed wilt and root rot and died after 15-20 days. Controls remained healthy. White mycelial colonies were formed over tissues of diseased LF and T and upon observation under the microscope, typical pear-shaped swellings were observed in slides prepared from newly obtained pure cultures from LF and T. Dematophora bunodes (formerly Rosellinia bunnodes) has a worldwide distribution and is well known as a polyphagous plant pathogen (Farr and Rossman, 2020) but has never been reported as a pathogen either of LF or T before in Brazil and worldwide. Its report on LF and T further expands an already large host-range and resolves the etiology of the disease on LF and T.

A fungus-eat-fungus world: Digitopodium, with particular reference to mycoparasites of the coffee leaf rust, Hemileia vastatrix.

Digitopodium hemileiae was described originally in 1930 as Cladosporium hemileiae; growing as a mycoparasite of the coffee leaf rust (CLR), Hemileia vastatrix, in a sample of diseased leaves of Coffea canephora collected in the Democratic Republic of Congo. No cultures from this material exist. More recently, the type material was re-examined and, based on morphological features, considered to be incorrectly placed in Cladosporium. The new genus Digitopodium was erected to accommodate this species. Interest in fungal antagonists of H. vastarix, as potential biocontrol agents of CLR, led to comprehensive surveys for mycoparasites, both in the African centre of origin of the rust, as well as in its South American exotic range. Among the rust specimens from Ethiopia, one was found to be colonized by a fungus congeneric with, and similar to, D. hemileiae. Pure cultures obtained from the Ethiopian material enabled a molecular study and for its phylogenetic position to be elucidated, based on DNA sequence data from the ITS and LSU regions. Molecular data showed that two members of the recently erected genus Hyalocladosporiella (Herpotrichiellaceae: Chaetothyriales) are congeneric with Digitopodium from Ethiopia and morphologically similar to both D. hemileiae and the two Ethiopian isolates. These isolates were found to be morphologically and genetically identical to H. tectonae, described previously from Brazil. Thus, species of Hyalocladosporiella are re-allocated to Digitopodium here; including D. tectonae, and a novel species, D. canescens, recently found in Brazil growing as a mycoparasite of Puccinia thaliae. The potential use of D. hemileiae and D. tectonae for classical biological control of CLR is discussed.

Bipolaris marantae sp. nov., A Novel Helminthosporoid Species Causing Foliage Blight of the Garden Plant Maranta leuconeura in Brazil.

A severe leaf spot, turning to foliage blight, was observed on leaves of Maranta leuconeura growing in a garden in Brazil (state of Rio de Janeiro) in 2015. A dematiaceous hyphomycete bearing a morphology typical of a helminthosporoid fungi was regularly found in association with diseased tissues. The fungus was isolated and pathogenicity was demonstrated through the completion of Koch's postulates. A morphology and molecular analysis led to the conclusion that the fungus belonged to the genus Bipolaris, which is characterized by having fusiform conidia, externally thickened and truncate hila and a bipolar pattern of germination. Additionally, homology of internal transcribed spacer and GAPDH sequences with sequences of other Bipolaris species, confirmed its generic placement. A phylogenetic study also indicated clearly that the fungus on M. leuconeura is phylogenetically distinct from related species of this genus, leading to the proposal of the new species Bipolaris marantae.