Aspergillus flavus from coffee in Cameroon: a non-aflatoxigenic endophytic isolate antagonistic to coffee leaf rust (Hemileia vastatrix).

AIMS: Elucidating the identity of an isolate of Aspergillus sp. obtained during searches for anti-coffee leaf rust (CLR) biocontrol agents, from healthy coffee berry samples, preliminarily verify whether it is an aflatoxin-producer, confirm its ability to grow as an endophyte in healthy coffee tissues and assess its biocontrol potential against CLR. METHODS AND RESULTS: One, among hundreds of fungal isolates fungus were obtained from healthy coffee tissues belonged to Aspergillus (isolate COAD 3307). A combination of morphology features and molecular analyses; including four regions-internal transcribed spacer, second-largest subunit of RNA polymerase (RPB2), ß-tubulin (BenA) and calmodulin (CAL)-identified COAD 3307 as Aspergillus flavus. Inoculations of healthy Coffea arabica with COAD 3307 confirmed its establishment as an endophyte in leaves, stems, and roots. Treatment of C. arabica plants by combinated applications of COAD 3307 on aerial parts and in the soil, significantly (P > .0001) reduced CLR severity as compared to controls. Thin-layer chromatography indicated that COAD 3307 is not an aflatoxin-producing isolate. In order to confirm this result, the extract was injected into high-performance liquid chromatography system equipped with a fluorescence detector, and no evidence of aflatoxin was found. CONCLUSIONS: COAD 3307 is an endophytic isolate of A. flavus-a species that has never been previously recorded as an endophyte of Coffea spp. It is a non-aflatoxin producing strain that has an anti-CLR effect and merits further evaluation as a biocontrol agent.

Filling a gap in the taxonomy of phyllachoroid fungi: Proposition of Neopolystigma, gen. nov., and the new family Neopolystigmataceae.

A Polystigma-like found on an herbaceous to shrubby species of Fabaceae (Andira humilis) in the Brazilian Cerrado was morphologically close to Polystigma pusillum, a leaf parasite on Andira inermis collected in Central America and Brazil. Phylogenetic analyses using a combination of the rDNA 28S, 18S, and internal transcribed spacer (ITS) regions placed both fungi in the Phyllachorales, and not in Polystigamataceae/Xylariales, where Polystigma species belong, and characteristically found on members of the Rosaceae, causing red leaf blotch containing bright-colored fungal stromata spread on the lesions. This disease prevails in orchards in the Northern Hemisphere, infecting Amygdalus, Cerasus, Padus, and Prunus species, but never in the Tropics. Polystigma species infecting other botanical families have been reallocated in different families, orders, and even classes in Ascomycota. In our phylogenetic analyses, the two species on Andira were allocated in Phyllachorales but separated in a well-supported cluster from Phyllachoraceae and Phaeochoraceae. In relation to Telimenaceae, the statistical support is not strong; however, considering that its type genus, Telimena, was never sequenced, we choose to accept Neopolystigma (type N. saraivae) as the type genus of a new family, Neopolystigmataceae. The sister species of N. saraivae, Polystigma pusillum found on A. inermis, was recombined into N. pusillum.

2,5-Diketopiperazines via Intramolecular N-Alkylation of Ugi Adducts: A Contribution to the Synthesis, Density Functional Theory Study, X-ray Characterization, and Potential Herbicide Application.

To investigate the herbicidal potential of 2,5-diketopiperazines (2,5-DKPs), we applied a known protocol to produce a series of 2,5-DKPs through intramolecular N-alkylation of Ugi adducts. However, the method was not successful for the cyclization of adducts presenting aromatic rings with some substituents at the ortho position. Results from DFT calculations showed that the presence of voluminous groups at the ortho position of a benzene ring results in destabilization of the transition structure. Lower activation enthalpies for the SN2-type cyclization of Ugi adducts were obtained when bromine, instead of a chlorine anion, is the leaving group, indicating that the activation enthalpy for the cyclization step controls the formation of the 2,5-DKP. Some Ugi adducts and 2,5-DKPs formed crystals with suitable qualities for single-crystal X-ray diffraction data collection. Phytotoxic damage of some 2,5-DKPs on leaves of the weed Euphorbia heterophylla did not differ from those caused by the commercial herbicide diquat.

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.

New species and records of Trichoderma isolated as mycoparasites and endophytes from cultivated and wild coffee in Africa.

A survey for species of the genus Trichoderma occurring as endophytes of Coffea, and as mycoparasites of coffee rusts (Hemileia), was undertaken in Africa; concentrating on Cameroon and Ethiopia. Ninety-four isolates of Trichoderma were obtained during this study: 76 as endophytes of healthy leaves, stems and berries and, 18 directly from colonized rust pustules. A phylogenetic analysis of all isolates used a combination of three genes: translation elongation factor-1α (tef1), rpb2 and cal for selected isolates. GCPSR criteria were used for the recognition of species; supported by morphological and cultural characters. The results reveal a previously unrecorded diversity of Trichoderma species endophytic in both wild and cultivated Coffea, and mycoparasitic on Hemileia rusts. Sixteen species were delimited, including four novel taxa which are described herein: T. botryosum, T. caeruloviride, T. lentissimum and T. pseudopyramidale. Two of these new species, T. botryosum and T. pseudopyramidale, constituted over 60% of the total isolations, predominantly from wild C. arabica in Ethiopian cloud forest. In sharp contrast, not a single isolate of Trichoderma was obtained using the same isolation protocol during a survey of coffee in four Brazilian states, suggesting the existence of a 'Trichoderma void' in the endophyte mycobiota of coffee outside of Africa. The potential use of these African Trichoderma isolates in classical biological control, either as endophytic bodyguards-to protect coffee plants from Hemileia vastatrix, the fungus causing coffee leaf rust (CLR)-or to reduce its impact through mycoparasitism, is discussed, with reference to the on-going CLR crisis in Central America.

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.

Moniliophthora perniciosa, the mushroom causing witches' broom disease of cacao: Insights into its taxonomy, ecology and host range in Brazil.

Witches' broom caused by Moniliophthora perniciosa is the main disease of cacao (Theobroma cacao) in Brazil. The fungus is known to occur on other host families and these populations have been addressed in the literature as biotypes: C (Malvaceae); H (Malpighiaceae); L (Bignoniaceae) and S (Solanaceae). No complete elucidation of the phylogenetic relationships of isolates obtained from this disparate host range appears in the literature. One member of H (ex Heteropterys acutifolia) has been described as a distinct species. But should other biotypes be also recognized as distinct taxa? In the present study, a survey yielding 24 isolates of M. perniciosa from ten hosts and covering a wide range of geographic regions in Brazil was undertaken. These isolates were compared with those from T. cacao using three DNA regions for the phylogenetic analyses: ITS, LSU and RPB1. Morphology was also examined. All isolates in this study were found to belong to M. perniciosa, including the population from H. acutifolia, formerly treated as Moniliophthora brasiliensis but reduced here to a synonym of M. perniciosa. This species ranged from pathogenic to a previously unreported occurrence as a non-pathogenic endophyte in the Atlantic rainforest tree Allophylus edulis (Sapindaceae). M. perniciosa was recorded on a range of solanaceous hosts (16 species) over a wide variety of ecosystems. The ecological and evolutionary significance of these novel findings are discussed.

Chocolate Under Threat from Old and New Cacao Diseases.

Theobroma cacao, the source of chocolate, is affected by destructive diseases wherever it is grown. Some diseases are endemic; however, as cacao was disseminated from the Amazon rain forest to new cultivation sites it encountered new pathogens. Two well-established diseases cause the greatest losses: black pod rot, caused by several species of Phytophthora, and witches' broom of cacao, caused by Moniliophthora perniciosa. Phytophthora megakarya causes the severest damage in the main cacao producing countries in West Africa, while P. palmivora causes significant losses globally. M. perniciosa is related to a sister basidiomycete species, M. roreri which causes frosty pod rot. These Moniliophthora species only occur in South and Central America, where they have significantly limited production since the beginnings of cacao cultivation. The basidiomycete Ceratobasidium theobromae causing vascular-streak dieback occurs only in South-East Asia and remains poorly understood. Cacao swollen shoot disease caused by Cacao swollen shoot virus is rapidly spreading in West Africa. This review presents contemporary research on the biology, taxonomy and genomics of what are often new-encounter pathogens, as well as the management of the diseases they cause.

Entomopathogenic fungi and their potential for the management of Aedes aegypti (Diptera: Culicidae) in the Americas

Classical biological control has been used extensively for the management of exotic weeds and agricultural pests, but never for alien insect vectors of medical importance. This simple but elegant control strategy involves the introduction of coevolved natural enemies from the centre of origin of the target alien species. Aedes aegypti - the primary vector of the dengue, yellow fever and Zika flaviviruses - is just such an invasive alien in the Americas where it arrived accidentally from its West African home during the slave trade. Here, we introduce the concept of exploiting entomopathogenic fungi from Africa for the classical biological control of Ae. aegypti in the Americas. Fungal pathogens attacking arthropods are ubiquitous in tropical forests and are important components in the natural balance of arthropod populations. They can produce a range of specialised spore forms, as well as inducing a variety of bizarre behaviours in their hosts, in order to maximise infection. The fungal groups recorded as specialised pathogens of mosquito hosts worldwide are described and discussed. We opine that similar fungal pathogens will be found attacking and manipulating Ae. aegypti in African forests and that these could be employed for an economic, environmentally-safe and long-term solution to the flavivirus pandemics in the Americas.

Entomopathogenic fungi and their potential for the management of Aedes aegypti (Diptera: Culicidae) in the Americas.

Classical biological control has been used extensively for the management of exotic weeds and agricultural pests, but never for alien insect vectors of medical importance. This simple but elegant control strategy involves the introduction of coevolved natural enemies from the centre of origin of the target alien species. Aedes aegypti - the primary vector of the dengue, yellow fever and Zika flaviviruses - is just such an invasive alien in the Americas where it arrived accidentally from its West African home during the slave trade. Here, we introduce the concept of exploiting entomopathogenic fungi from Africa for the classical biological control of Ae. aegypti in the Americas. Fungal pathogens attacking arthropods are ubiquitous in tropical forests and are important components in the natural balance of arthropod populations. They can produce a range of specialised spore forms, as well as inducing a variety of bizarre behaviours in their hosts, in order to maximise infection. The fungal groups recorded as specialised pathogens of mosquito hosts worldwide are described and discussed. We opine that similar fungal pathogens will be found attacking and manipulating Ae. aegypti in African forests and that these could be employed for an economic, environmentally-safe and long-term solution to the flavivirus pandemics in the Americas.

Recognition of endophytic Trichoderma species by leaf-cutting ants and their potential in a Trojan-horse management strategy.

Interactions between leaf-cutting ants, their fungal symbiont (Leucoagaricus) and the endophytic fungi within the vegetation they carry into their colonies are still poorly understood. If endophytes antagonistic to Leucoagaricus were found in plant material being carried by these ants, then this might indicate a potential mechanism for plants to defend themselves from leaf-cutter attack. In addition, it could offer possibilities for the management of these important Neotropical pests. Here, we show that, for Atta sexdens rubropilosa, there was a significantly greater incidence of Trichoderma species in the vegetation removed from the nests-and deposited around the entrances-than in that being transported into the nests. In a no-choice test, Trichoderma-infested rice was taken into the nest, with deleterious effects on both the fungal gardens and ant survival. The endophytic ability of selected strains of Trichoderma was also confirmed, following their inoculation and subsequent reisolation from seedlings of eucalyptus. These results indicate that endophytic fungi which pose a threat to ant fungal gardens through their antagonistic traits, such as Trichoderma, have the potential to act as bodyguards of their plant hosts and thus might be employed in a Trojan-horse strategy to mitigate the negative impact of leaf-cutting ants in both agriculture and silviculture in the Neotropics. We posit that the ants would detect and evict such 'malign' endophytes-artificially inoculated into vulnerable crops-during the quality-control process within the nest, and, moreover, that the foraging ants may then be deterred from further harvesting of 'Trichoderma-enriched' plants.

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.

A new Inocyclus species (Parmulariaceae) on the neotropical fern Pleopeltis astrolepis.

During a survey for fungal pathogens associated with ferns in Brazil, a tar spot-causing fungus was found on fronds of Pleopeltis astrolepis. This was recognised as belonging to Inocyclus (Parmulariaceae). After comparison with other species in the genus, it was concluded that the fungus on P. astrolepis is a new species, described here as Inocyclus angularis sp. nov.

Rhagadolobiopsis, a new genus of Parmulariaceae from Brazil with a description of the ontogeny of its ascomata.

During a survey of the mycobiota of selected Brazilian ferns we discovered a new genus of Parmulariaceae causing tar spot-like symptoms on leaves of Thelypteris serrata (Thelypteridaceae). The new genus and species, decribed as Rhagadolobiopsis thelypteridis, differs from morphologically similar species of Rhagadolobium in possessing colorless, aseptate ascospores and a hymenial gel that does not become blue with iodine. In addition this is the first record of a fungus on T. serrata, and the first Parmulariaceae recorded on a member of the Thelypteridaceae. The ontogeny of the ascomata is described and illustrated here for the first time for the Parmulariaceae.

Yet more "weeds" in the garden: fungal novelties from nests of leaf-cutting ants.

BACKGROUND: Symbiotic relationships modulate the evolution of living organisms in all levels of biological organization. A notable example of symbiosis is that of attine ants (Attini; Formicidae: Hymenoptera) and their fungal cultivars (Lepiotaceae and Pterulaceae; Agaricales: Basidiomycota). In recent years, this mutualism has emerged as a model system for studying coevolution, speciation, and multitrophic interactions. Ubiquitous in this ant-fungal symbiosis is the "weedy" fungus Escovopsis (Hypocreales: Ascomycota), known only as a mycoparasite of attine fungal gardens. Despite interest in its biology, ecology and molecular phylogeny--noting, especially, the high genetic diversity encountered--which has led to a steady flow of publications over the past decade, only two species of Escovopsis have formally been described. METHODS AND RESULTS: We sampled from fungal gardens and garden waste (middens) of nests of the leaf-cutting ant genus Acromyrmex in a remnant of subtropical Atlantic rainforest in Minas Gerais, Brazil. In culture, distinct morphotypes of Escovopsis sensu lato were recognized. Using both morphological and molecular analyses, three new species of Escovopsis were identified. These are described and illustrated herein--E. lentecrescens, E. microspora, and E. moelleri--together with a re-description of the genus and the type species, E. weberi. The new genus Escovopsioides is erected for a fourth morphotype. We identify, for the first time, a mechanism for horizontal transmission via middens. CONCLUSIONS: The present study makes a start at assigning names and formal descriptions to these specific fungal parasites of attine nests. Based on the results of this exploratory and geographically-restricted survey, we expect there to be many more species of the genus Escovopsis and its relatives associated with nests of both the lower and higher Attini throughout their neotropical range, as suggested in previous studies.

Passalora stromatica sp. nov. associated with leaf spots of Tithonia diversifolia in Brazil.

Tithonia diversifolia, a member of Compositae native to Central America that produces showy sunflower-like flowers, became an invasive weed in other continents after it was introduced as an ornamental. Little is known about fungal pathogens infecting this plant. Knowledge of its mycobiota is of interest for future biocontrol programmes for T. diversifolia. In Brazil, a cercosporoid hyphomycete was found associated with intense leaf-spotting of this plant. Based on morphological and molecular data it was recognized as representing a new species of Passalora, and the name Passalora stromatica sp.nov. is introduced here for this taxon. This fungus is described and illustrated herein. It is possible that this fungus is playing a role in Brazil in reducing the invasiveness of T. diversifolia as, contrarily to what has been reported for countries in Africa and Asia, it remains mostly as a garden escape or rural plant in Brazil.

Pilidiella tibouchinae sp. nov. associated with foliage blight of Tibouchina granulosa (quaresmeira) in Brazil.

Tibouchina granulosa (Melastomataceae), Brazilian glorytree (Brazilian common name - quaresmeira), a common tree of the Atlantic Forest of Brazil, is widely used as an ornamental for its violet or pink blossoms. Little is known about fungal diseases affecting this species, although these represent a known limitation for its cultivation in nurseries. Among these there is a foliage blight that occurs in combination with distortion of branch apices and die-back. A consistent association of a species of Pilidiella with the diseased tissues was observed. The fungus was isolated in pure culture and based on its morphology and DNA phylogeny, we conclude that it represents a new species, for which the name Pilidiella tibouchinae is introduced.

Reappraisal and neotypification of Phyllachora feijoae.

Acca sellowiana (Myrtaceae), feijoa (in Brazil, goiaba da serra), is a native southern South America tree that produces edible fruits which, although only occasionally cultivated in South America, became a significant fruit crop in New Zealand. Recently, during surveys for fungal pathogens of feijoa in southern Brazil, several plants were found bearing tar-spot symptoms caused by a species of Phyllachora. A literature search enabled us to identify the fungus as Phyllachora feijoae, a little-known species originally described in the 19(th) century by H. Rehm and later transferred to the genus Catacauma. The name Catacauma feijoae, although now regarded as a later synonym of P. feijoae is still mistakenly in use (as, for instance, in the Brazilian list of fungi on plants). The type specimen was most probably deposited in the Botanisches Garten und Museum Berlin-Dahlem (B) and lost or destroyed during World War II, and could not be located. The recent recollection of abundant material of this fungus in the vicinity of Pelotas (Rio Grande do Sul, Brazil) allowed its re-examination and neotypification. Phyllachora feijoae is also illustrated here for the first time.