Phylogenetic and Pathogenic Evidence Reveals Novel Host-Pathogen Interactions between Species of Lasiodiplodia and Citrus latifolia Dieback Disease in Southern Mexico.

Mexico ranks second in the world for Persian lime (Citrus latifolia) exports, making it the principal citrus exporter within the national citrus industry, exporting over 600,000 tons per year. However, diseases are the main factor reducing production, resulting in significant economic losses. Among these diseases, fungal diseases like dieback, caused by species of Lasiodiplodia, are an emerging issue in Persian lime. Symptoms include gummosis, twig and branch dieback, cankers, the necrosis of bark and wood, fruit mummification, and tree decline. The aim of this study was to investigate the occurrence and pathogenicity of the fungal species associated with twig and branch dieback, cankers, and decline of Persian lime trees in southern Mexico, and to elucidate the current status of the Lasiodiplodia species causing the disease in Mexico. During June, July, and August of 2023, a total of the 9229 Persian lime trees were inspected across 230 hectares of Persian lime orchards in southern Mexico, and symptoms of the disease were detected in 48.78% of the trees. Branches from 30 of these Persian lime trees were collected. Fungal isolates were obtained, resulting in a collection of 40 strains. The isolates were characterized molecularly and phylogenetically through the partial regions of four loci: the internal transcribed spacer region (ITS), the ß-tubulin gene (tub2), the translation elongation factor 1-alpha gene (tef1-α), and the DNA-directed RNA polymerase II second largest subunit (rpb2). Additionally, pathogenicity was assessed, successfully completing Koch's postulates on both detached Persian lime branches and certified 18-month-old Persian lime plants. Through multilocus molecular phylogenetic identification, pathogenicity, and virulence tests, five species were identified as causal agents: L. iraniensis, L. lignicola, L. mexicanensis, L. pseudotheobromae, and L. theobromae. This study demonstrates that in southern Mexico, at least five species of the genus Lasiodiplodia are responsible for dieback in Persian lime. Additionally, this is the first report of L. lignicola and L. mexicanensis as causal agents of the disease in citrus, indicating novel host interactions between species of Lasiodiplodia and C. latifolia.

Population Genetic Analysis of Fusarium decemcellulare, a Guaraná Pathogen, Reveals High Genetic Diversity in the Amazonas State, Brazil.

Guaraná is indigenous to the Brazilian Amazon where it has cultural and agroeconomic significance. However, its cultivation is constrained by a disease termed oversprouting of guaraná caused by Fusarium decemcellulare, with yield losses reaching as high as 100%. The disease can affect different parts of the plant, causing floral hypertrophy and hyperplasia, stem galls, and oversprouting of vegetative buds. To date, no study has been conducted characterizing the genetic diversity and population structure of this pathogen. Here, we report genetic diversity and genetic structure among 224 isolates from eight guaraná production areas of Amazonas State, Brazil, that were genotyped using a set of 10 inter-simple-sequence repeat (ISSR) markers. Despite moderate gene diversity (Hexp = 0.21 to 0.32), genotypic diversity was at or near maximum (223 multilocus genotypes among 224 isolates). Population genetic analysis of the 10 ISSR marker fragments with STRUCTURE software identified two populations designated C1 and C2 within the F. decemcellulare collection from the eight sites. Likewise, UPGMA hierarchical clustering and discriminant analysis of principal components of the strains from guaraná resolved these same two groups. Analysis of molecular variance demonstrated that 71% of genetic diversity occurred within the C1 and C2 populations. A pairwise comparison of sampling sites for both genetic populations revealed that 59 of 66 were differentiated from one another (P < 0.05), and high and significant gene flow was detected only between sampling sites assigned to the same genetic population. The presence of MAT1-1 and MAT1-2 strains, in conjunction with the high genotypic diversity and no significant linkage disequilibrium, suggests that each population of F. decemcellulare might be undergoing sexual reproduction. Isolation by distance was not observed (R2 = 0.02885, P > 0.05), which suggests that human-mediated movement of seedlings may have played a role in shaping the F. decemcellulare genetic structure in Amazonas State, Brazil.

Fusarium chaquense, sp. nov, a novel type A trichothecene-producing species from native grasses in a wetland ecosystem in Argentina.

The Chaco wetland is among the most biologically diverse regions in Argentina. In collections of fungi from asymptomatic native grasses (Poaceae) from the wetlands, we identified isolates of Fusarium that were morphologically similar to F. armeniacum, but distinct from it by their production of abundant microconidia. All the isolates had identical, or nearly identical, partial sequences of TEF1 and RPB2. But they were distinct from reference sequences from F. armeniacum and Fusarium species closely related to it. Phylogenetic analysis of 34 full-length housekeeping gene sequences retrieved from whole genome sequences of three Chaco wetland isolates, 29 genes resolved the isolates as an exclusive clade within the F. sambucinum species complex. Based on results of the morphological and phylogenetic analysis, we concluded that the Chaco wetland isolates are a distinct and novel species, herein described as Fusarium chaquense, sp. nov., which is closely related to F. armeniacum. F. chaquense in culture can produce the trichothecenes T-2 and HT-2 toxin, neosolaniol, diacetoxyscirpenol, and monoacetoxyscirpenol, as well as beauvericin and the pigment aurofusarin. Genome sequence analysis also revealed the presence of three previously described loci required for trichothecene biosynthesis. This research represents the first study of Fusarium in a natural ecosystem in Argentina.

Weeds Harbor Fusarium Species that Cause Malformation Disease of Economically Important Trees in Western Mexico.

Mango malformation disease (MMD) caused by Fusarium spp. is an important limiting factor in most production areas worldwide. Fusarium mexicanum and F. pseudocircinatum have been reported as causing MMD in Mexico. These two pathogens also cause a similar disease in Swietenia macrophylla (big-leaf mahogany malformation disease) in central western Mexico, and F. pseudocircinatum was recently reported as causing malformation disease in Tabebuia rosea (rosy trumpet) in the same region. These studies suggest that additional plant species, including weeds, might be hosts of these pathogens. The role that weed hosts might have in the disease cycle is unknown. The objectives of this work were to recover Fusarium isolates from understory vegetation in mango orchards with MMD, identify the Fusarium isolates through DNA sequence data, and determine whether F. mexicanum is capable of inducing disease in the weedy legume Senna uniflora (oneleaf senna). Additional objectives in this work were to compare Fusarium isolates recovered from weeds and mango trees in the same orchards by characterizing their phylogenetic relationships, assessing in vitro production of mycotoxins, and identifying their mating type idiomorph. A total of 59 Fusarium isolates from five species complexes were recovered from apical and lateral buds from four weed species. Two of the species within the F. fujikuroi species complex are known to cause MMD in Mexico. Trichothecene production was detected in five isolates, including F. sulawense and F. irregulare in the F. incarnatum-equiseti species complex and F. boothii in the F. sambucinum species complex. Both mating types were present among mango and weed isolates. This is the first report of herbaceous hosts harboring Fusarium species that cause mango malformation in Mexico. The information provided should prove valuable for further study of the epidemiological role of weeds in MMD and help manage the disease.

Malformation Disease in Tabebuia rosea (Rosy Trumpet) Caused by Fusarium pseudocircinatum in Mexico.

Tabebuia rosea (rosy trumpet) is an economically important neotropical tree in Mexico that is highly valued for the quality of its wood, which is used for furniture, crafts, and packing, and for its use as an ornamental and shade tree in parks and gardens. During surveys conducted in the lower Balsas River Basin region in the states of Guerrero and Michoacán, symptoms of floral malformation were detected in T. rosea trees. The main objectives of this study were to describe this new disease, to determine its causal agent, and to identify it using DNA sequence data. A second set of objectives was to analyze the phylogenetic relationship of the causal agent to Fusarium spp. associated with Swietenia macrophylla trees with malformation surveyed in the same region and to compare mycotoxin production and the mating type idiomorphs of fusaria recovered from T. rosea and S. macrophylla. Tabebuia rosea showed malformed inflorescences with multiple tightly curled shoots and shortened internodes. A total of 31 Fusarium isolates recovered from symptomatic T. rosea (n = 20) and S. macrophylla (n = 11) trees were identified by molecular analysis as Fusarium pseudocircinatum. Pathogenicity tests showed that isolates of F. pseudocircinatum recovered from T. rosea induced malformation in inoculated T. rosea seedlings. Eighteen F. pseudocircinatum isolates were tested for their ability to produce mycotoxins and other secondary metabolites. Moniliformin, fusaric acid, bikaverin, beauvericin, aurofusarin. and 8-O-methylbostrycoidin were produced by at least one strain of the 18 isolates tested. A multiplex PCR assay for mating type idiomorph revealed that 22 F. pseudocircinatum isolates were MAT1-1 and that 9 were MAT1-2. Here, we report a new disease of T. rosea in Mexico caused by F. pseudocircinatum.

Fusarium xyrophilum, sp. nov., a member of the Fusarium fujikuroi species complex recovered from pseudoflowers on yellow-eyed grass (Xyris spp.) from Guyana.

We report on the discovery and characterization of a novel Fusarium species that produced yellow-orange pseudoflowers on Xyris spp. (yellow-eyed grass; Xyridaceae) growing in the savannas of the Pakaraima Mountains of western Guyana. The petaloid fungal structures produced on infected plants mimic host flowers in gross morphology. Molecular phylogenetic analyses of full-length RPB1 (RNA polymerase largest subunit), RPB2 (RNA polymerase second largest subunit), and TEF1 (elongation factor 1-α) DNA sequences mined from genome sequences resolved the fungus, described herein as F. xyrophilum, sp. nov., as sister to F. pseudocircinatum within the African clade of the F. fujikuroi species complex. Results of a polymerase chain reaction (PCR) assay for mating type idiomorph revealed that single-conidial isolates of F. xyrophilum had only one of the MAT idiomorphs (MAT1-1 or MAT1-2), which suggests that the fungus may have a heterothallic sexual reproductive mode. BLASTn searches of whole-genome sequence of three strains of F. xyrophilum indicated that it has the genetic potential to produce secondary metabolites, including phytohormones, pigments, and mycotoxins. However, a polyketide-derived pigment, 8-O-methylbostrycoidin, was the only metabolite detected in cracked maize kernel cultures. When grown on carnation leaf agar, F. xyrophilum is phenotypically distinct from other described Fusarium species in that it produces aseptate microconidia on erect indeterminate synnemata that are up to 2 mm tall and it does not produce multiseptate macroconidia.

Taxonomy and phylogeny of Macrolepiota: two new species from Brazil.

Macrolepiota is a poorly known genus in the Neotropics. In order to increase knowledge about this group, we collected specimens from the Atlantic Forest in southern and northeastern Brazil. Macrolepiota cyanolamellata and M. sabulosa from subtropical and tropical regions, respectively, are proposed as new species. We performed molecular phylogenetic analyses of the nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS) and the combined data set ITS + nuclear large subunit rDNA (28S) + RNA polymerase II second largest (RPB2), as well as morphological analyses. Two lineages with unique morphotypes were found. The species proposed were strongly supported as the sister lineage closely related to M. clelandii and M. subcitrophylla. Detailed descriptions and illustrations of their macro- and microscopic characters are provided.

In vitro susceptibility and multilocus sequence typing of Fusarium isolates causing keratitis.

Fungal keratitis is recognized as a significant cause of ocular morbidity and blindness especially in developing countries. In this study, we aimed to present the molecular identification and susceptibility of Fusarium isolates causing fungal keratitis in a university hospital in southern Brazil. The samples were identified using the second largest subunit of the RNA polymerase gene (RPB2) and the translation elongation factor 1-alpha (TEF1), while the antifungal susceptibility was tested by the broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) methodology. The majority of the isolates belonged to the Fusarium solani species complex (F. solani, F. keratoplasticum and F. falciforme) and Fusarium oxysporum species complex. Antifungal susceptibility has shown that amphotericin B and natamycin were the most effective antifungals across all isolates, followed by voriconazole. Variation among Fusarium complexes in their antifungal sensitivities was observed in our study. The identification of Fusarium species from human samples is important not only from an epidemiological viewpoint, but also for choosing the appropriate antifungal agent for difficult-to-treat Fusarium infections such as keratitis.

Multigene phylogeny of the Phallales (Phallomycetidae, Agaricomycetes) focusing on some previously unrepresented genera.

Phylogenetic relationships within the Phallales were estimated via combined sequences: nuclear ribosomal large subunit (LSU), second largest subunit of RNA polymerase (rpb2), and mitochondrial ATPase subunit 6 (atp6). The ingroup is represented by 62 taxa comprising 18 genera and 44 species, including members of the Clathraceae, Claustulaceae, Gastrosporiaceae, Lysuraceae, Phallaceae and Protophallaceae. Sixty-one new sequences were generated, including tropical and subtropical taxa. This is one of the first studies discussing the phylogenetic placement of Abrachium, Aseroë, Blumenavia, Gastrosporium, Jansia and Xylophallus. Gastrosporiaceae was demonstrated to be sister to Phallaceae and an emended description of the order is presented. Aseroë was demonstrated to be polyphyletic and as a result, A. arachnoidea is transferred to Lysurus.

Two new species of Lactarius associated with Alnus acuminata subsp. arguta in Mexico.

In pure stands of Alnus acuminata subsp. arguta trees from Sierra Norte de Puebla (central Mexico) two undescribed ectomycorrhizal species of Lactarius were discovered. Distinction of the two new species is based on morphological characters and supported with phylogenetic analyses of the nuclear ribosomal DNA ITS region and part of the gene that encodes for the second largest subunit of RNA polymerase II (rpb2). The phylogenies inferred recovered the two species in different clades strongly supported by posterior probabilities and bootstrap values. The new Lactarius species are recognized as part of the assemblage of ectomycorrhizal fungi associated with Alnus acuminata. Information about these taxa includes the morphological variation achieved along 16 monitories 2010-2013. Descriptions are provided. They are accompanied by photos including SEM photomicrographs of basidiospores and information on differences between them and other related taxa from Europe and the United States.