Fusarium: more than a node or a foot-shaped basal cell

Recent publications have argued that there are potentially serious consequences for researchers in recognising distinct genera in the terminal fusarioid clade of the family Nectriaceae. Thus, an alternate hypothesis, namely a very broad concept of the genus Fusarium was proposed. In doing so, however, a significant body of data that supports distinct genera in Nectriaceae based on morphology, biology, and phylogeny is disregarded. A DNA phylogeny based on 19 orthologous protein-coding genes was presented to support a very broad concept of Fusarium at the F1 node in Nectriaceae. Here, we demonstrate that re-analyses of this dataset show that all 19 genes support the F3 node that represents Fusarium sensu stricto as defined by F. sambucinum (sexual morph synonym Gibberella pulicaris). The backbone of the phylogeny is resolved by the concatenated alignment, but only six of the 19 genes fully support the F1 node, representing the broad circumscription of Fusarium. Furthermore, a re-analysis of the concatenated dataset revealed alternate topologies in different phylogenetic algorithms, highlighting the deep divergence and unresolved placement of various Nectriaceae lineages proposed as members of Fusarium. Species of Fusarium s. str. are characterised by Gibberella sexual morphs, asexual morphs with thin- or thick-walled macroconidia that have variously shaped apical and basal cells, and trichothecene mycotoxin production, which separates them from other fusarioid genera. Here we show that the Wollenweber concept of Fusarium presently accounts for 20 segregate genera with clear-cut synapomorphic traits, and that fusarioid macroconidia represent a character that has been gained or lost multiple times throughout Nectriaceae. Thus, the very broad circumscription of Fusarium is blurry and without apparent synapomorphies, and does not include all genera with fusarium-like macroconidia, which are spread throughout Nectriaceae (e.g., Cosmosporella, Macroconia, Microcera). In this study four new genera are introduced, along with 18 new species and 16 new combinations. These names convey information about relationships, morphology, and ecological preference that would otherwise be lost in a broader definition of Fusarium. To assist users to correctly identify fusarioid genera and species, we introduce a new online identification database, Fusarioid-ID, accessible at www.fusarium.org. The database comprises partial sequences from multiple genes commonly used to identify fusarioid taxa (act1, CaM, his3, rpb1, rpb2, tef1, tub2, ITS, and LSU). In this paper, we also present a nomenclator of names that have been introduced in Fusarium up to January 2021 as well as their current status, types, and diagnostic DNA barcode data. In this study, researchers from 46 countries, representing taxonomists, plant pathologists, medical mycologists, quarantine officials, regulatory agencies, and students, strongly support the application and use of a more precisely delimited Fusarium (= Gibberella) concept to accommodate taxa from the robust monophyletic node F3 on the basis of a well-defined and unique combination of morphological and biochemical features. This F3 node includes, among others, species of the F. fujikuroi, F. incarnatum-equiseti, F. oxysporum, and F. sambucinum species complexes, but not species of Bisifusarium [F. dimerum species complex (SC)], Cyanonectria (F. buxicola SC), Geejayessia (F. staphyleae SC), Neocosmospora (F. solani SC) or Rectifusarium (F. ventricosum SC). The present study represents the first step to generating a new online monograph of Fusarium and allied fusarioid genera (www.fusarium.org)

Fig 2 -

Dynamics of Microdochium bolleyi colonization in (A) wheat and (B) Brachypodium distachyon over time analyzed by qPCR. Statistically significant differences are indicated by asterixis (post hoc Tukey’s test, P < 0.05).</p

Fig 1 -

Chlamydospores of Microdochium bolleyi in wheat leaf sheaths (A, B) and comparison of seed coats of non-inoculated (C) and inoculated (D) Brachypodium distachyon. Scale bar = 100 μm.</p

The efficiency (E) of the reaction was determined using a dilution series of DNA from the <i>Microdochium bolleyi</i> isolate (UPOC-FUN-253) with primers MbqITS.

The efficiency (E) of the reaction was determined using a dilution series of DNA from the Microdochium bolleyi isolate (UPOC-FUN-253) with primers MbqITS.</p

Resulting Cq values after qPCR with five primer pairs.

Resulting Cq values after qPCR with five primer pairs.</p

Colonization of wheat tissues by light microscopy and qPCR.

Colonization of wheat tissues by light microscopy and qPCR.</p

Primer pairs used in the study.

Names, sequences of forward and reverse primers, publication sources of primer pairs, and gene functions are listed. (DOCX)</p

Schema of the sampling method.

Microdochium bolleyi is a fungal endophyte of cereals and grasses proposed as an ideal model organism for studying plant-endophyte interactions. A qPCR-based diagnostic assay was developed to detect M. bolleyi in wheat and Brachypodium distachyon tissues using the species-specific primers MbqITS derived from the ITS of the ribosomal gene. Specificity was tested against 20 fungal organisms associated with barley and wheat. Colonization dynamics, endophyte distribution in the plant, and potential of the seed transmission were analyzed in the wheat and model plant B. distachyon. The colonization of plants by endophyte starts from the germinating seed, where the seed coats are first strongly colonized, then the endophyte spreads to the adjacent parts, crown, roots near the crown, and basal parts of the stem. While in the lower distal parts of roots, the concentration of M. bolleyi DNA did not change significantly in successive samplings (30, 60, 90, 120, and 150 days after inoculation), there was a significant increase over time in the roots 1 cm under crown, crowns and stem bases. The endophyte reaches the higher parts of the base (2–4 cm above the crown) 90 days after sowing in wheat and 150 days in B. distachyon. The endophyte does not reach both host species’ leaves, peduncles, and ears. Regarding the potential for seed transmission, endophyte was not detected in harvested grains of plants with heavily colonized roots. Plants grown from seeds derived from parental plants heavily colonized by endophyte did not exhibit any presence of the endophyte, so transmission by seeds was not confirmed. The course of colonization dynamics and distribution in the plant was similar for both hosts tested, with two differences: the base of the wheat stem was colonized earlier, but B. distachyon was occupied more intensively and abundantly than wheat. Thus, the designed species-specific primers could detect and quantify the endophyte in planta.</div

Fungal species used in the analysis with the MbqITS primers designed in this study.

A positive reaction result is indicated by a Cq lower than 30. The Cq levels in the table are the average of three replicates.</p

Seed transfer analysis by qPCR.

Microdochium bolleyi is a fungal endophyte of cereals and grasses proposed as an ideal model organism for studying plant-endophyte interactions. A qPCR-based diagnostic assay was developed to detect M. bolleyi in wheat and Brachypodium distachyon tissues using the species-specific primers MbqITS derived from the ITS of the ribosomal gene. Specificity was tested against 20 fungal organisms associated with barley and wheat. Colonization dynamics, endophyte distribution in the plant, and potential of the seed transmission were analyzed in the wheat and model plant B. distachyon. The colonization of plants by endophyte starts from the germinating seed, where the seed coats are first strongly colonized, then the endophyte spreads to the adjacent parts, crown, roots near the crown, and basal parts of the stem. While in the lower distal parts of roots, the concentration of M. bolleyi DNA did not change significantly in successive samplings (30, 60, 90, 120, and 150 days after inoculation), there was a significant increase over time in the roots 1 cm under crown, crowns and stem bases. The endophyte reaches the higher parts of the base (2–4 cm above the crown) 90 days after sowing in wheat and 150 days in B. distachyon. The endophyte does not reach both host species’ leaves, peduncles, and ears. Regarding the potential for seed transmission, endophyte was not detected in harvested grains of plants with heavily colonized roots. Plants grown from seeds derived from parental plants heavily colonized by endophyte did not exhibit any presence of the endophyte, so transmission by seeds was not confirmed. The course of colonization dynamics and distribution in the plant was similar for both hosts tested, with two differences: the base of the wheat stem was colonized earlier, but B. distachyon was occupied more intensively and abundantly than wheat. Thus, the designed species-specific primers could detect and quantify the endophyte in planta.</div