Patulin contamination of hard apple cider by Paecilomyces niveus and other postharvest apple pathogens: Assessing risk factors.

Hard apple cider is considered to be a low-risk product for food spoilage and mycotoxin contamination due to its alcoholic nature and associated food sanitation measures. However, the thermotolerant mycotoxin-producing fungus Paecilomyces niveus may pose a significant threat to hard cider producers. P. niveus is known to infect apples (Malus xdomestica), and previous research indicates that it can survive thermal processing and contaminate finished apple juice with the mycotoxin patulin. To determine if hard apple cider is susceptible to a similar spoilage phenomenon, cider apples were infected with P. niveus or one of three patulin-producing Penicillium species and the infected fruits underwent benchtop fermentation. Cider was made with lab inoculated Dabinett and Medaille d'Or apple cultivars, and patulin was quantified before and after fermentation. Results show that all four fungi can infect cider apples and produce patulin, some of which is lost during fermentation. Only P. niveus was able to actively grow throughout the fermentation process. To determine if apple cider can be treated to hinder P. niveus growth, selected industry-grade sanitation measures were tested, including chemical preservatives and pasteurization. High concentrations of preservatives inhibited P. niveus growth, but apple cider flash pasteurization was not found to significantly impact spore germination. This study confirms that hard apple cider is susceptible to fungal-mediated spoilage and patulin contamination. P. niveus is an important concern for hard apple cider producers due to its demonstrated thermotolerance, survival in fermentative environments, and resistance to sanitation measures.

The Early Terrestrial Fungal Lineage of Conidiobolus-Transition from Saprotroph to Parasitic Lifestyle.

Fungi of the Conidiobolus group belong to the family Ancylistaceae (Entomophthorales, Entomophthoromycotina, Zoopagomycota) and include over 70 predominantly saprotrophic species in four similar and closely related genera, that were separated phylogenetically recently. Entomopathogenic fungi of the genus Batkoa are very close morphologically to the Conidiobolus species. Their thalli share similar morphology, and they produce ballistic conidia like closely related entomopathogenic Entomophthoraceae. Ballistic conidia are traditionally considered as an efficient tool in the pathogenic process and an important adaptation to the parasitic lifestyle. Our study aims to reconstruct the phylogeny of this fungal group using molecular and genomic data, ancestral lifestyle and morphological features of the conidiobolus-like group and the direction of their evolution. Based on phylogenetic analysis, some species previously in the family Conidiobolaceae are placed in the new families Capillidiaceae and Neoconidiobolaceae, which each include one genus, and the Conidiobolaceae now includes three genera. Intermediate between the conidiobolus-like groups and Entomophthoraceae, species in the distinct Batkoa clade now belong in the family Batkoaceae. Parasitism evolved several times in the Conidiobolus group and Ancestral State Reconstruction suggests that the evolution of ballistic conidia preceded the evolution of the parasitic lifestyle.

Susceptibility of Rosaceous Pome and Stone Fruits to Postharvest Rot by Paecilomyces niveus.

Paecilomyces rot of apples is a postharvest disease caused by Paecilomyces niveus, a problematic spoiling agent of fruit juices and derivatives. Processing fruits infected with Paecilomyces rot can lead to juices contaminated with P. niveus ascospores. These ascospores are heat resistant and may survive food processing and germinate in finished products. Because the fungus produces the mycotoxin patulin, juice spoilage by P. niveus is an important health hazard. Little is known about the disease biology and control mechanisms of this recently described postharvest disease. The range of fruit products contaminated by P. niveus and patulin led us to hypothesize that the host range of Paecilomyces rot is broader than previously thought. Following Koch's postulates, we determined that multiple untested rosaceous fruits and popular apple cultivars are susceptible to Paecilomyces rot infection and that these infected fruits contain significant levels of patulin. We also observed that two closely related food spoiling fungi, Paecilomyces fulvus and Paecilomyces variotti, were unable to infect, cause symptoms in, or grow in wounded fruits. Therefore, we challenge the assumption that P. niveus spoilage inoculum is introduced to foods solely through environmental sources, and we show that other economically important rosaceous fruits, peaches, pears, sweet cherries, and sour cherries, are susceptible to infection and can also be sources of spoilage inoculum. Our results highlight the unique abilities of P. niveus to infect a variety of fruits, produce patulin, and form resistant spores capable of spoiling normally shelf-stable products.

Evolutionary relationships among Massospora spp. (Entomophthorales), obligate pathogens of cicadas.

The fungal genus Massospora (Zoopagomycota: Entomophthorales) includes more than a dozen obligate, sexually transmissible pathogenic species that infect cicadas (Hemiptera) worldwide. At least two species are known to produce psychoactive compounds during infection, which has garnered considerable interest for this enigmatic genus. As with many Entomophthorales, the evolutionary relationships and host associations of Massospora spp. are not well understood. The acquisition of M. diceroproctae from Arizona, M. tettigatis from Chile, and M. platypediae from California and Colorado provided an opportunity to conduct molecular phylogenetic analyses and morphological studies to investigate whether these fungi represent a monophyletic group and delimit species boundaries. In a three-locus phylogenetic analysis including the D1-D2 domains of the nuclear 28S rRNA gene (28S), elongation factor 1 alpha-like (EFL), and beta-tubulin (BTUB), Massospora was resolved in a strongly supported monophyletic group containing four well-supported genealogically exclusive lineages, based on two of three methods of phylogenetic inference. There was incongruence among the single-gene trees: two methods of phylogenetic inference recovered trees with either the same topology as the three-gene concatenated tree (EFL) or a basal polytomy (28S, BTUB). Massospora levispora and M. platypediae isolates formed a single lineage in all analyses and are synonymized here as M. levispora. Massospora diceroproctae was sister to M. cicadina in all three single-gene trees and on an extremely long branch relative to the other Massospora, and even the outgroup taxa, which may reflect an accelerated rate of molecular evolution and/or incomplete taxon sampling. The results of the morphological study presented here indicate that spore measurements may not be phylogenetically or diagnostically informative. Despite recent advances in understanding the ecology of Massospora, much about its host range and diversity remains unexplored. The emerging phylogenetic framework can provide a foundation for exploring coevolutionary relationships with cicada hosts and the evolution of behavior-altering compounds.

Psychoactive plant- and mushroom-associated alkaloids from two behavior modifying cicada pathogens.

Entomopathogenic fungi routinely kill their hosts before releasing infectious spores, but a few species keep insects alive while sporulating, which enhances dispersal. Transcriptomics- and metabolomics-based studies of entomopathogens with post-mortem dissemination from their parasitized hosts have unraveled infection processes and host responses. However, the mechanisms underlying active spore transmission by Entomophthoralean fungi in living insects remain elusive. Here we report the discovery, through metabolomics, of the plant-associated amphetamine, cathinone, in four Massospora cicadina-infected periodical cicada populations, and the mushroom-associated tryptamine, psilocybin, in annual cicadas infected with Massospora platypediae or Massospora levispora, which likely represent a single fungal species. The absence of some fungal enzymes necessary for cathinone and psilocybin biosynthesis along with the inability to detect intermediate metabolites or gene orthologs are consistent with possibly novel biosynthesis pathways in Massospora. The neurogenic activities of these compounds suggest the extended phenotype of Massospora that modifies cicada behavior to maximize dissemination is chemically-induced.

Nature Abhors a Vacuum: Highly Diverse Mechanisms Enable Spoilage Fungi to Disperse, Survive, and Propagate in Commercially Processed and Preserved Foods.

Fungal spoilage in processed foods remains a challenge for food manufacturers despite the increasing availability of diverse processing and formulation strategies used to control foodborne microorganisms. Physiological features of yeasts and molds contribute to their tolerance to thermal processing, acidity, desiccation, and oxygen and nutrient limitations. These features variably include growth form, cell wall structure, cytoplasmic composition, cell membrane-bound proteins, and secretion of secondary metabolites. Collectively, these mechanisms contribute to the ability of fungi to disperse, survive, and propagate in highly restrictive food environments. The diversity of fungal growth and survival mechanisms has resulted in organisms adapted to nearly all food environments; although, only a small subset of fungi are particularly suited for spoilage of a given product. The relationship between the individual physiology and metabolic capabilities of a yeast or mold and the product's specific physicochemical attributes and processing history determines spoilage potential. Explicit characterization of the fungal features responsible for this extremotolerance contributes to more targeted and effective control strategies.

Paecilomyces niveus: Pathogenicity in the Orchard and Sensitivity to Three Fungicides.

Paecilomyces rot of apples is a postharvest disease caused by the thermotolerant fungus Paecilomyces niveus (Byssochlamys nivea). The etiology of disease and the activity of fungicides against P. niveus are not yet well understood. This study evaluated the ability of P. niveus to infect 'Gala' apples growing in a conventionally managed orchard. In addition, the sensitivity of P. niveus isolates to postharvest fungicides difenoconazole, fludioxonil, and pyrimethanil was characterized for isolates from both agricultural and nonagricultural environments. Apples were wounded and mock-inoculated or inoculated with P. niveus in early July. At the time of harvest, 8 weeks after wounding, the inoculated apples had significantly larger lesions than mock-inoculated apples (P < 0.005). The average diameter of lesions on wound-inoculated apples was 11.17 mm ± 6.82 SD, while the average diameter of mock-inoculated lesions was 3.34 mm ± 1.85 SD. Disease symptoms in the orchard were similar to postharvest symptoms of Paecilomyces rot. Symptoms included a brown, flattened, circular lesion with faint concentric rings. The necrosis of mesocarp was firm and roughly U-shaped. Baseline isolates of P. niveus, from nonagricultural environments, were used to determine the effective fungicide dose at which growth was inhibited by 50% (EC50). Furthermore, fungicide sensitivity of P. niveus isolates was examined using relative growth assays at the mean baseline EC50 values to compare baseline isolates with isolates obtained from commercial apple orchards where they were likely exposed to fungicides. Among the exposed isolates, reduced sensitivity to all fungicides was observed, but significant differences between baseline and exposed isolates were only observed with fludioxonil (P < 0.0001). This is the first report demonstrating that P. niveus can infect apples that are wound-inoculated in the orchard and that isolates from agricultural environments are less sensitive to common fungicides, especially fludioxonil. This finding may have implications for the control of this postharvest diseases. Whether natural infections of apples by P. niveus is initiated in the orchard or during postharvest has yet to be determined.

Draft Genome Sequence of the Patulin-Producing Fungus Paecilomyces niveus Strain CO7.

Paecilomyces niveus is an extremotolerant fungus with surprising powers to survive high temperatures and infect apples and aphids. These abilities make it a formidable enemy in food and agricultural environments. In addition, it produces patulin, the most significant mycotoxin in apples.

Sea salts as a potential source of food spoilage fungi.

Production of sea salt begins with evaporation of sea water in shallow pools called salterns, and ends with the harvest and packing of salts. This process provides many opportunities for fungal contamination. This study aimed to determine whether finished salts contain viable fungi that have the potential to cause spoilage when sea salt is used as a food ingredient by isolating fungi on a medium that simulated salted food with a lowered water activity (0.95 aw). The viable filamentous fungi from seven commercial salts were quantified and identified by DNA sequencing, and the fungal communities in different salts were compared. Every sea salt tested contained viable fungi, in concentrations ranging from 0.07 to 1.71 colony-forming units per gram of salt. In total, 85 fungi were isolated representing seven genera. One or more species of the most abundant genera, Aspergillus, Cladosporium, and Penicillium was found in every salt. Many species found in this study have been previously isolated from low water activity environments, including salterns and foods. We conclude that sea salts contain many fungi that have potential to cause food spoilage as well as some that may be mycotoxigenic.

Paecilomyces Rot: A New Apple Disease.

Paecilomyces niveus is an important food spoilage fungus that survives thermal processing in fruit products, where it produces the mycotoxin patulin. Spoilage of products has been attributed to soil contamination; however, little is known about the ecology of this organism. In this study, orchard soils and culled apple fruit were surveyed and the ability of P. niveus to infect apple was tested on two popular apple varieties. P. niveus was found in 34% of sampled orchard soils from across New York. Completing Koch's postulates, P. niveus was demonstrated to cause postharvest disease in Gala and Golden Delicious apple. Symptoms of this disease, named Paecilomyces rot, resemble several other apple diseases, including black rot, bitter rot, and bull's-eye rot. External symptoms of Paecilomyces rot include brown, circular, concentrically ringed lesions, with an internal rot that is firm and cone-shaped. Both Gala and Golden Delicious apple fruit inoculated with P. niveus developed lesions ≥43 mm in size at 22 days after inoculation. There is some evidence that the size of lesions and rate of infection differ between Gala and Golden Delicious, which may indicate differing resistance to P. niveus. This work shows that P. niveus is common in New York orchard soil and can cause a novel postharvest fruit disease. Whether infected fruit can serve as an overlooked source of inoculum in heat-processed apple products requires further study.

The first entomophthoralean killing millipedes, Arthrophaga myriapodina n. gen. n. sp., causes climbing before host death.

A new species and genus of entomophthoralean fungus, Arthrophaga myriapodina kills polydesmid millipedes. This species was first seen over a century ago but never described. It is the first millipede pathogen known from the order Entomophthorales, species of which are best known as pathogens of a wide diversity of insects. The fungus induces pre-death climbing behavior in its hosts, enabling the fungus to broadcast its forcibly-discharged conidia from a high vantage, which presumably increases the fitness of the fungus. Study of herbarium specimens and photographic discoveries on the internet suggest the fungus occurs widely in eastern North America.

Speciation of a tropical fungal species pair following transoceanic dispersal.

This study focuses on a pair of fungal species, Moelleriella libera and M. raciborskii (Ascomycota: Clavicipitaceae) from the neotropics and paleotropics, respectively, that are phenotypically nearly indistinguishable. Molecular analyses based on DNA sequences from RNA polymerase II subunit 2 (RPB2), translation elongation factor 1-alpha (EF1-alpha) and beta-tubulin genes confirm that they are recently derived sister species. Speciation appears to have followed an historical transoceanic dispersal event. Models of population structure and migration from TCS, IM, and coalescent-based analyses suggest there is little gene flow between the two species. The direction of dispersal, investigated using the progression rule and coalescent-based gene genealogies, was likely from the New World to the Old World.

Variability of the mitochondrial SSU rDNA of Nomuraea species and other entomopathogenic fungi from hypocreales.

Hypocrealean arthropod pathogenic fungi have profound impact on the regulation of agricultural and medical pests. However, until now the genetic and phylogenetic relationships among species have not been clarified, such studies could clarify host specificity relationships and define species boundaries. Our purpose was to compare the sequences of the mitochondrial SSU rDNA fragments from several mitosporic entomopathogenic Hypocreales to infer relationships among them and to evaluate the possibility to use these sequences as species diagnostic tool in addition to the more commonly studied sequences of nuclear SSU rDNA. The SSU mt-rDNA proved to be useful to help in differentiation of species inside several genera. Clusters obtained with Parsimony, Bayesian, and Maximum Likelihood analyses were congruent with a new classification of the Clavicipitaceae (Sung et al. Stud Mycol. 2007;57:5-59) in which the anamorphic genera Nomuraea and Metarhizium species remain in the Clavicipitaceae and Isaria species sequenced here are assigned to the family Cordycipitaceae. Mitochondrial genomic information indicates the same general pattern of relationships demonstrated by nuclear gene sequences.

Diversity and Aggressiveness of Rhizoctonia solani and Rhizoctonia-like Fungi on Vegetables in New York.

Vegetable growers in New York, especially those growing table beets, have recently observed that the corn rotation is no longer effective in suppressing diseases caused by Rhizoctonia solani and Rhizoctonia-like fungi. To investigate this problem, 68 isolates of Rhizoctonia solani and Rhizoctonia-like fungi infecting vegetables in New York were isolated, characterized, and their pathogenicity on corn determined. Sequence analysis of the rDNA internal transcribed spacer region inferred 26 isolates to belong to R. solani anastomosis group (AG) 2-2 and 19 isolates to belong to AG 4. Remaining isolates belonged to AG 1, AG 2-1, AG 5, AG 11, Ceratobasidium AG (CAG) 2, CAG 6, and Waitea circinata var. zeae. This is a first report of AG 11 and W. circinata var. zeae recovered from naturally infected vegetables in New York. Pathogenicity tests on corn showed that the majority of isolates are pathogenic on corn, and isolates belonging to AG 2-2, AG 5, and AG 11 exhibited high aggressiveness. These results suggest that certain strains of R. solani and Rhizoctonia-like fungi infecting vegetables in New York have acquired the ability to infect corn. In addition, snap bean was inoculated with seven isolates exhibiting low to high aggressiveness on corn, and a correlation between aggressiveness on corn and snap bean was observed.

A taxonomic revision of the insect biocontrol fungus Aschersonia aleyrodis, its allies with white stromata and their Hypocrella sexual states.

A revision of a monophyletic group of Hypocrella species and their Aschersonia anamorphs with white effuse stromata is presented. In addition to taxon descriptions, distributions, and nomenclature, a synoptic key and a molecular phylogenetic analysis are also provided. A new holomorph, Hypocrella rhombispora sp. nov., is described. This study presents a revisionary treatment of Aschersonia aleyrodis (teleomorph: Hypocrella libera) and its allies. These fungi parasitize whiteflies and are promising candidates for whitefly biological control. Four species of Aschersonia and their Hypocrella teleomorphs are treated in detail: Aschersonia aleyrodis/Hypocrella libera; A. andropogonis/H. andropogonis, A. placenta/H. raciborskii, and A. sp./H. rhombispora sp. nov. A synoptic key including these and six other morphologically similar species is presented to facilitate identification in the field and laboratory. Phylogenetic analyses of partial DNA sequences from three genes (LSU, mtSSU, and RPB2) suggest that Aschersonia species with effuse white stromata form a monophyletic group of whitefly pathogens. Phylogenetically informative characters in the group include the colour and shape of the stromata, the arrangement of tubercles containing perithecia, the arrangement of conidial masses on the stromata, and the shape of conidia and part spores.

A new species of Hypocrella, H. macrostroma, and its phylogenetic relationships to other species with large stromata.

Two specimens of a new species of Hypocrella with large stromata were collected in Bolivia and Costa Rica. The morphology of the new species, H. macrostroma sp. nov., was compared with that of other species with large stromata, i.e. H. africana, H. gaertneriana, and H. schizostachyi. In addition, phylogenetic analyses of partial sequences from three genes, large subunit nuclear ribosomal DNA (LSU), translation elongation factor 1-alpha (EF1-alpha), and RNA polymerase II subunit 1 (RPB1), were conducted to determine the relationships of the new species to other species of Hypocrella/Aschersonia. Phylogenetic analyses show that H. macrostroma belongs to a strongly supported clade that includes H. africana, H. schizostachyi, and Aschersonia insperata, whereas other Hypocrella species belong to two sister clades. Hypocrella macrostroma is described and illustrated, and a lectotype is designated for H. gaertneriana.

Hypocrella zhongdongii sp. nov., the teleomorph of aschersonia incrassata.

A new Hypocrella species with white pulvinate stromata collected in Puerto Rico and Costa Rica is described as H. zhongdongii sp. nov. Morphological and molecular evidence confirms that the new species of Hypocrella is the teleomorph of Aschersonia incrassata. It most closely resembles H. andropogonis; both A. incrassata and A. andropogonis are common yellow-spored species. The relationships of H. zhongdongii with other species in the genus are elucidated through phylogenetic analyses of three different genetic loci (LSU, RPB2, and mtSSU). Our analysis also sheds light on current subgeneric concepts in Aschersonia, in which the presence or absence of conidiomatal paraphyses is a major character to separate the genus into two subgenera. The present phylogenetic tree suggests that paraphyses have been lost or gained multiple times during evolutionary history, and do not define monophyletic groups.

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.

What's in a name? Aschersonia insperata: a new pleoanamorphic fungus with characteristics of Aschersonia and Hirsutella.

A new anamorphic species from a Philippine tropical forest occurs as reddish-orange to orange, tuberculate stromata on unidentified homopteran larvae, and produces both Aschersonia and Hirsutella-like synanamorphs. A molecular phylogenetic analysis was conducted to determine the most appropriate generic placement for this fungus. Based on its phylogenetic relationships, a comparison of the complexity and persistence of each anamorph, and the speculated relevance of each synanamorph to survival, we describe the new fungus as Aschersonia insperata sp. nov.

The genus Podocrella and its nematode-killing anamorph Harposporium.

Several genera are described in the literature as having morphology similar to the clavicipitaceous genus Podocrella, viz. Atricordyceps, Ophiocordyceps, Wakefieldiomyces and "Cordyceps" peltata. These genera have capitate-stipitate stromata that gradually expand into a horizontally flattened fertile head that is dark, has strongly protruding perithecia and asci containing eight multiseptate filiform ascospores. These ascospores disarticulate at the middle septum to form two lanceolate multiseptate part-as-cospores. In this study several specimens of the above-mentioned genera, including the types, were examined to determine whether they are congeneric with Podocrella. This study also reveals the connection of Podocrella to its anamorph genus, Harposporium, and its relationship to several other clavicipitaceous genera, based on cultural data and large subunit nuclear ribosomal DNA (LSU) sequences. Nematode predation of the Harposporium anamorph of P. peltata is demonstrated. The results show Podocrella and selected Harposporium LSU sequences form a monophyletic group and that this clade is closely related to Aschersonia. A new species of Podocrella from Costa Rica, P fusca, is described, new combinations made for P. peltata and P. harposporifera, and a key to the known species is presented.