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.

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.

Exploiting exotic pathogens as mycoherbicides against invasive alien weeds: Japanese knotweed as a case study.

Plant species have been introduced increasingly into non-native ranges, where many have become exotic weeds with adverse impacts on native ecosystems, as well as on farming and other livelihoods. In biological control, the classical or inoculative approach is the one most commonly used for the management of invasive alien weeds and is based on the use of co-evolved natural enemies from the native range to control the invasive weed. Typically, the inundative or mycoherbicide approach targets problematic weeds using local plant pathogens that, in the case of introduced species, have 'jumped' onto the exotic host. The leaf-spot fungus, Mycosphaerella polygoni-cuspidati, co-evolved with its host, Reynoutria (Fallopia) japonica (Japanese knotweed), in Japan and has a unique history of being investigated both as a classical biological control agent and a mycoherbicide against this highly invasive weed in the United Kingdom and North America. Here, we highlight our research on M. polygoni-cuspidati as part of a biological control programme for Japanese knotweed and review the potential of mycoherbicides using exotic pathogens for the management of invasive alien weeds. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Cordyceps cateniannulata: An endophyte of coffee, a parasite of coffee leaf rust and a pathogen of coffee pests.

Here, we report on a Cordyceps species entering into a multi-trophic, multi-kingdom association. Cordyceps cateniannulata, isolated from the stem of wild Coffea arabica in Ethiopia, is shown to function as an endophyte, a mycoparasite and an entomopathogen. A detailed polyphasic taxonomic study, including a multilocus phylogenetic analysis, confirmed its identity. An emended description of C. cateniannulata is provided herein. Previously, this species was known as a pathogen of various insect hosts in both the Old and New World. The endophytic status of C. cateniannulata was confirmed by re-isolating it from inoculated coffee plants. Inoculation studies have further shown that C. cateniannulata is a mycoparasite of Hemileia vastatrix, as well as an entomopathogen of major coffee pests; infecting and killing Hypothenemus hampei and Leucoptera coffeella. This is the first record of C. cateniannulata from Africa, as well as an endophyte and a mycoparasite. The implications for its use as a biocontrol agent are discussed.

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.

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.

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.

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.

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.

Are the microcyclic rust species Puccinia melampodii and Puccinia xanthii conspecific?

The taxonomic relationship between two microcyclic rust species, Puccinia melampodii and Puccinia xanthii, recorded from a number of Asteraceae hosts, was explored by comparing teliospore morphology, host specificity and sequence data. Teliospore morphology varied between and within individual rust accessions but, in general, teliospores of P. xanthii were larger than those of P. melampodii. Field observations and cross-inoculation greenhouse studies showed that P. melampodii accessions were highly specific to their original host species and unable to establish compatible interactions with related Asteraceae species. Though small amounts of genetic variation were detected between P. melampodii and P. xanthii accessions, the ITS and TEF phylogenetic analyses comprising several other rust species grouped these accessions within a well supported clade. Our data indicate that despite differences between P. xanthii and P. melampodii accessions these rusts cannot readily be separated at the species level, supporting earlier taxonomic conclusions and the recent proposal to group them into a morphospecies. Based on host-specificity data, the name Puccinia xanthii var. parthenii-hysterophorae is proposed to accommodate all Mesoamerican records of P. melampodii associated with the host Parthenium hysterophorus.

Moniliophthora roreri, causal agent of cacao frosty pod rot.

Taxonomy: Moniliophthora roreri (Cif.) H.C. Evans et al. ; Phylum Basidiomycota; Class Agaricomycetes; Order Agaricales; Family Marasmiaceae; Genus Moniliophthora. Biology: Moniliophthora roreri attacks Theobroma and Herrania species causing frosty pod rot. Theobroma cacao (cacao) is the host of major economic concern. Moniliophthora roreri is a hemibiotroph with a long biotrophic phase (45-90 days). Spore masses, of apparent asexual origin, are produced on the pod surface after initiation of the necrotrophic phase. Spores are spread by wind, rain and human activity. Symptoms of the biotrophic phase can include necrotic flecks and, in some cases, pod malformation, but pods otherwise remain asymptomatic. Relationship to Moniliophthora perniciosa: Moniliophthora roreri and Moniliophthora perniciosa, causal agent of witches' broom disease of cacao, are closely related. Their genomes are similar, including many of the genes they carry which are considered to be important in the disease process. Moniliophthora perniciosa, also a hemibiotroph, has a typical basidiomycete lifestyle and morphology, forming clamp connections and producing mushrooms. Basidiospores infect meristematic tissues including flower cushions, stem tips and pods. Moniliophthora roreri does not form clamp connections or mushrooms and infects pods only. Both pathogens are limited to the Western Hemisphere and are a threat to cacao production around the world. Agronomic importance: Disease losses caused by frosty pod rot can reach 90% and result in field abandonment. Moniliophthora roreri remains in the invasive phase in the Western Hemisphere, not having reached Brazil, some islands within the Caribbean and a few specific regions within otherwise invaded countries. DISEASE MANAGEMENT: The disease can be managed by a combination of cultural (for example, maintenance of tree height and removal of infected pods) and chemical methods. These methods benefit from regional application, but can be cost prohibitive. Breeding for disease resistance offers the greatest potential for frosty pod rot management and new tolerant materials are becoming available.

Deciphering the biology of Cryptophyllachora eurasiatica gen. et sp. nov., an often cryptic pathogen of an allergenic weed, Ambrosia artemisiifolia.

A little known, unculturable ascomycete, referred to as Phyllachora ambrosiae, can destroy the inflorescences of Ambrosia artemisiifolia, an invasive agricultural weed and producer of highly allergenic pollen. The fungus often remains undetectable in ragweed populations. This work was conducted to understand its origin and pathogenesis, a prerequisite to consider its potential as a biocontrol agent. The methods used included light and transmission electron microscopy, nrDNA sequencing, phylogenetic analyses, artificial inoculations, and the examination of old herbarium and recent field specimens from Hungary, Korea, Ukraine and USA. The Eurasian and the North American specimens of this fungus were to represent two distinct, although closely related lineages that were only distantly related to other lineages within the Ascomycota. Consequently, we describe a new genus that includes Cryptophyllachora eurasiatica gen. et sp. nov. and C. ambrosiae comb. nov., respectively. The pathogenesis of C. eurasiatica was shown in A. artemisiifolia. No evidence was found for either seed-borne transmission or systemic infection. Two hypotheses were developed to explain the interaction between C. eurasiatica and A. artemisiifolia: (i) as yet undetected seed-borne transmissions and latent, systemic infections; or (ii) alternative hosts.

Cacao diseases-the trilogy revisited.

ABSTRACT This paper reviews the significant advances by the diseases themselves, as well as by the scientists, in the intervening period since the disease trilogy was first delimited in 1989. The impact of these diseases, black pod, witches' broom, and frosty pod rot, has increased dramatically. In addition, there have been radical changes in the taxonomic profiles of these pathogens, which have been based on both traditional (morphological, cytological) and modern (molecular) approaches. Black pod is caused by a complex of Phytophthora species, in which P. palmivora still is the most important worldwide. However, recent invasion of the principal cacao-growing countries of West Africa by the more virulent P. megakarya has been cause for concern. The latter evolved in the ancient forests straddling the Cameroon-Nigerian border as a primary coloniser of fallen fruit. Conversely, frosty pod rot, caused by Moniliophthora roreri, and witches' broom, caused by M. (Crinipellis) perniciosa, both neotropical diseases, are hemibiotrophic, coevolved pathogens. Respectively, M. roreri arose on Theobroma gileri in submontane forests on the north-western slopes of the Andes, whereas M. perniciosa developed as a complex of pathotypes with a considerably wider geographic and host range within South America; the cacao pathotype evolved on that host in the Amazon basin. The inter-relationships of these vicariant species and their recent spread are discussed, together with control strategies.

Putting the waste out: a proposed mechanism for transmission of the mycoparasite Escovopsis between leafcutter ant colonies.

The attine ant system is a remarkable example of symbiosis. An antagonistic partner within this system is the fungal parasite Escovopsis, a genus specific to the fungal gardens of the Attini. Escovopsis parasitizes the Leucoagaricus symbiont that leaf-cutting ants (Acromyrmex, Atta) have been farming over the past 8-12 Myr. However, it has been a puzzle how Escovopsis reaches its host. During a seasonal survey of nests of Acromyrmex subterraneus subterraneus in Atlantic rainforest in Brazil, Escovopsis was detected in all the sampled fungal garden waste tips or middens (n = 111). Middens were built strategically; always below the nest entrances. Here, we report the first evidence of a putative mechanism for horizontal transmission of Escovopsis between attine colonies. It is posited that leaf-cutting ants pick up the spores from soil and litter during foraging and vector the mycoparasite between attine colonies. Field and laboratory experiments, using At. laevigata and Ac. subterraneus subterraneus, confirm that Escovopsis spores are phoretic, and have an inbuilt dormancy, broken by the presence of their Leucoagaricus host. However, in the coevolutionary arms race, Atta ants may lose out-despite most species in the genus investing in a more advanced waste disposal system-due to the insanitary habits of their Acromyrmex neighbours.

Ant-infecting Ophiocordyceps genomes reveal a high diversity of potential behavioral manipulation genes and a possible major role for enterotoxins.

Much can be gained from revealing the mechanisms fungal entomopathogens employ. Especially intriguing are fungal parasites that manipulate insect behavior because, presumably, they secrete a wealth of bioactive compounds. To gain more insight into their strategies, we compared the genomes of five ant-infecting Ophiocordyceps species from three species complexes. These species were collected across three continents, from five different ant species in which they induce different levels of manipulation. A considerable number of (small) secreted and pathogenicity-related proteins were only found in these ant-manipulating Ophiocordyceps species, and not in other ascomycetes. However, few of those proteins were conserved among them, suggesting that several different methods of behavior modification have evolved. This is further supported by a relatively fast evolution of previously reported candidate manipulation genes associated with biting behavior. Moreover, secondary metabolite clusters, activated during biting behavior, appeared conserved within a species complex, but not beyond. The independent co-evolution between these manipulating parasites and their respective hosts might thus have led to rather diverse strategies to alter behavior. Our data indicate that specialized, secreted enterotoxins may play a major role in one of these strategies.

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.

The biology and taxonomy of rust fungi associated with the neotropical vine Mikania micrantha, a major invasive weed in Asia.

Three microcyclic rust species were collected during surveys of the perennial asteraceous vine Mikania micrantha (Eupatorieae: Asteraceae) throughout its native range in the Neotropics but were absent in its invasive range in Asia. The commonest species, Puccinia spegazzinii with brown telioid telia, occurred wherever M. micrantha was found in South and Central America including the Caribbean island of Trinidad. Dietelia portoricensis, with occasional vestigial spermogonia and grayish-white to pale yellow columnar aecioid telia, was collected only in Costa Rica; while D. mesoamericana sp. nov., apparently restricted to Mesoamerica, can be distinguished by its abundant, yellowish-orange, fertile spermogonia, yellow to pale brown telial columns, larger teliospores, and 4-spored rather than 2-spored metabasidia. The fact that all three species share a fundamentally similar symptomatology suggests a common origin.

Regiocrella, a new entomopathogenic genus with a pycnidial anamorph and its phylogenetic placement in the Clavicipitaceae.

A new genus, Regiocrella, is described with two species, R. camerunensis and R. sinensis, based on specimens collected in Cameroon and China. Both species are parasitic on scale insects (Coccidae, Homoptera). Morphological and molecular evidence place the new genus in the Clavicipitaceae (Hypocreales), despite its combination of characters that are atypical of that family; Regiocrella is characterized by having perithecia partly immersed in a subiculum, noncapitate asci, unicellular fusiform ascospores and pycnidial-acervular conidiomata. The two new species, R. camerunensis and R. sinensis, are distinguished based on ascospore and perithecium size. Morphological characters were evaluated and compared to other genera in the Clavicipitaceae, especially those parasitic on scale insects or with pycnidial-acervular anamorphs or synanamorphs (i.e. Aschersonia, Ephelis or Sphacelia): Atkinsonella, Balansia, Claviceps, Epichlöe, Hypocrella, Myriogenospora and Neoclaviceps. The phylogenetic relationships of Regiocrella were examined with three gene loci: large subunit nuclear ribosomal DNA (LSU), translation elongation factor 1-alpha (TEF), and RNA polymerase II subunit 1 (RPB1). The results of this study confirm that Regiocrella is distinct from other genera in the Clavicipitaceae and that its two species form a monophyletic group. Regiocrella is shown to be closely related to the scale insect pathogen Hypocrella and the plant-associated genera Balansia, Claviceps, Epichlöe, Myriogenospora and Neoclaviceps. This study also provides insights into the evolution of pycnidial-acervular conidiomata and scale insect parasitism within the Clavicipitaceae. Plant-associated genera form a monophyletic group correlated with Clavicipitaceae subfamily Clavicipitoideae sensu Diehl. We also demonstrate that scale insect parasites have multiple evolutionary origins within the family and genera with pycnidial-acervular anamorphs or synanamorphs have a single origin.