The m6 A reader MhYTP2 negatively modulates apple Glomerella leaf spot resistance by binding to and degrading MdRGA2L mRNA.

Glomerella leaf spot (GLS), caused by the fungal pathogen Colletotrichum fructicola, significantly threatens apple production. Some resistances to plant disease are mediated by the accumulation of nucleotide-binding site and leucine-rich repeat (NBS-LRR) proteins that are encoded by a major class of plant disease resistance genes (R genes). However, the R genes that confer resistance to GLS in apple remain largely unclear. Malus hupehensis YT521-B homology domain-containing protein 2 (MhYTP2) was identified as an N6 -methyladenosine RNA methylation (m6 A) modified RNA reader in our previous study. However, whether MhYTP2 binds to mRNAs without m6 A RNA modifications remains unknown. In this study, we discovered that MhYTP2 exerts both m6 A-dependent and -independent functions by analysing previously obtained RNA immunoprecipitation sequencing results. The overexpression of MhYTP2 significantly reduced the resistance of apple to GLS and down-regulated the transcript levels of some R genes whose transcripts do not contain m6 A modifications. Further analysis indicated that MhYTP2 binds to and reduces the stability of MdRGA2L mRNA. MdRGA2L positively regulates resistance to GLS by activating salicylic acid signalling. Our findings revealed that MhYTP2 plays an essential role in the regulation of resistance to GLS and identified a promising R gene, MdRGA2L, for use in developing apple cultivars with GLS resistance.

Comparative transcriptome analysis reveals significant differences in gene expression between pathogens of apple Glomerella leaf spot and apple bitter rot.

BACKGROUND: Apple Glomerella leaf spot (GLS) and apple bitter rot (ABR) are two devastating foliar and fruit diseases on apples. The different symptoms of GLS and ABR could be related to different transcriptome patterns. Thus, the objectives of this study were to compare the transcriptome profiles of Colletotrichum gloeosporioides species complex isolates GC20190701, FL180903, and FL180906, the pathogen of GLS and ABR, and to evaluate the involvement of the genes on pathogenicity. RESULTS: A relatively large difference was discovered between the GLS-isolate GC20190701 and ABR-isolates FL180903, FL180906, and quite many differential expression genes associated with pathogenicity were revealed. The DEGs between the GLS- and ABR-isolate were significantly enriched in GO terms of secondary metabolites, however, the categories of degradation of various cell wall components did not. Many genes associated with secondary metabolism were revealed. A total of 17 Cytochrome P450s (CYP), 11 of which were up-regulated while six were down-regulated, and five up-regulated methyltransferase genes were discovered. The genes associated with the secretion of extracellular enzymes and melanin accumulation were up-regulated. Four genes associated with the degradation of the host cell wall, three genes involved in the degradation of cellulose, and one gene involved in the degradation of xylan were revealed and all up-regulated. In addition, genes involved in melanin syntheses, such as tyrosinase and glucosyltransferase, were highly up-regulated. CONCLUSIONS: The penetration ability, pathogenicity of GLS-isolate was greater than that of ABR-isolate, which might indicate that GLS-isolate originated from ABR-isolates by mutation. These results contributed to highlighting the importance to investigate such DEGs between GLS- and ABR-isolate in depth.

Phylogenetic relationships of Phaeochorellaparinarii and recognition of a new family, Phaeochorellaceae (Diaporthales).

Species of Phaeochorella are biotrophic leaf parasites with a tropical distribution, traditionally accepted in the family Phyllachoraceae, Phyllachorales in classifications based on morphological characters. Phylogenetic evidence presented here resolves the relationship of Phaeochorella within the Sordariomycetes, based on a multilocus analysis of partial nuc rDNA large subunit (28S) and internal transcribed spacers (ITS1-5.8S-ITS2 = ITS), the DNA-directed RNA polymerase II second largest subunit (RPB2), and the translation elongation factor 1-α (TEF1-α) gene. Phylogenetic analyses indicate that Phaeochorella belongs to the Diaporthales rather than the Phyllachorales. Phaeochorella parinarii, the type species of the genus, present on native hosts from the Brazilian Cerrado, forms a unique clade with a species of Phaeoappendicospora with high support. Thus, a new family, Phaeochorellaceae, Diaporthales, including both genera, is herein proposed. With the exception of P. parinarii and P. zonata, all other species in Phaeochorella (P. artocarpi, P. ciliata, P. machaerii) were excluded from the genus.

New neotropical species of Phyllachorales based on molecular, morphological, and ecological data.

Species of tropical tar spot fungi (Phyllachorales, Ascomycota) are obligate biotrophic plant parasitic fungi associated with living leaves of a wide range of families of host plants, mainly in tropical and subtropical regions. In this study, samples of tropical tar spot fungi were collected in forests in Costa Rica and Panamá. To identify taxa, we used morphology and information on host plants and combined multigene phylogeny of four genes: the large subunit nuclear ribosomal DNA (28S rDNA), the small subunit nuclear ribosomal DNA (18S rDNA), the complete internal transcribed spacer region of ribosomal DNA (nuc rDNA ITS1-5.8S-ITS2; ITS), and the translation elongation factor 1-α (tef1). Here we propose one new species in the genus Camarotella and eight new species in Telimena with their morphological descriptions, illustrations, and sequence data. The newly described species are Camarotella licaniae on Licania arborea (Chrysobalanaceae) and in the genus Telimena: T. billiae on Billia rosea (Sapindaceae), T. drymoniae on Drymonia multiflora (Gesneriaceae), T. hydrangeae on Hydrangea sp. (Hydrangeaceae), T. miravallensis on Symplocos panamensis (Symplocaceae), T. protii on Protium sp. (Burseraceae), T. rinoreae on Rinorea sp. (Violaceae), T. semialarii on Semialarium mexicanum (Celastraceae), and T. triseptata on Tapirira mexicana (Anacardiaceae). The new name Telimena nitens on Schlegelia brachyanta (Schlegeliaceae) is presented and 10 species of Phyllachora are transferred to Telimena, leading to the new combinations T. canarii, T. galavisii, T. insueta, T. ruelliae, T. scutiformis, T. serjaniicola, T. spicatae, T. subrepens, T. symploci, and T. symplocicola. Additionally, revisions of tar spot fungi on host families Burseraceae, Sapindaceae, and Symplocaceae are provided, and four new synonyms are proposed.

ITS phylogeny and taxonomy of Phyllachora species on native Myrtaceae from the Brazilian Cerrado.

Nine Phyllachora species found on hosts belonging to the family Myrtaceae native to the Brazilian Cerrado were described and illustrated. We sequenced nuc rDNA internal transcribed spacer barcode regions for representatives of seven species and conducted phylogenetic analyses, which provided strong support for four new species that we describe as Phyllachora cerradensis, P. ermidensis, P. furnasensis, and P. myrciariae. Catacauma nigerrimum was recombined into P. nigerrima, and a key to the common Phyllachora species on myrtaceous hosts from the Brazilian Cerrado was also included.

High level expression of Glomerella cingulata cutinase in dense cultures of Pichia pastoris grown under fed-batch conditions.

A Pichia pastoris transformant carrying the cutinase cDNA of Glomerella cingulata was over-expressed in a 5L bioreactor (2.0L working volume) under fed-batch conditions. Bioreactor experiments rely on varying selected parameters in repeated rounds of optimisation: here these included duration of induction, pH and temperature. Highest cell densities (320gL(-1) wet cell weight) with a cutinase production of 3800mgL(-1) and an activity of 434UmL(-1) were achieved 24h after induction with methanol in basal salt medium (at pH 5 and 28°C). Characterisation of the cutinase showed that it was stable between pH 6 and pH 11, had an optimum pH of 8.0 and retained activity for 30min at 50°C (optimum temperature 25°C).The preferred substrates of G. cingulata cutinase were the medium- to long-chain ρ-nitrophenyl esters of ρ-nitrophenylcaprylate (C8), ρ-nitrophenyllaurate (C12) and ρ-nitrophenylmyristate (C14), with the highest catalytic efficiency, kcat/Km of 7.7±0.7mM(-1)s(-1) for ρ-nitrophenylcaprylate. Microscopic analyses showed that the G. cingulata cutinase was also capable of depolymerising the high molecular weight synthetic polyester, polyethylene terephthalate.

Characterization of Glomerella strains recovered from anthracnose lesions on common bean plants in Brazil.

Anthracnose caused by Colletotrichum lindemuthianum is an important disease of common bean, resulting in major economic losses worldwide. Genetic diversity of the C. lindemuthianum population contributes to its ability to adapt rapidly to new sources of host resistance. The origin of this diversity is unknown, but sexual recombination, via the Glomerella teleomorph, is one possibility. This study tested the hypothesis that Glomerella strains that are frequently recovered from bean anthracnose lesions represent the teleomorph of C. lindemuthianum. A large collection of Glomerella isolates could be separated into two groups based on phylogenetic analysis, morphology, and pathogenicity to beans. Both groups were unrelated to C. lindemuthianum. One group clustered with the C. gloeosporioides species complex and produced mild symptoms on bean tissues. The other group, which belonged to a clade that included the cucurbit anthracnose pathogen C. magna, caused no symptoms. Individual ascospores recovered from Glomerella perithecia gave rise to either fertile (perithecial) or infertile (conidial) colonies. Some pairings of perithecial and conidial strains resulted in induced homothallism in the conidial partner, while others led to apparent heterothallic matings. Pairings involving two perithecial, or two conidial, colonies produced neither outcome. Conidia efficiently formed conidial anastomosis tubes (CATs), but ascospores never formed CATs. The Glomerella strains formed appressoria and hyphae on the plant surface, but did not penetrate or form infection structures within the tissues. Their behavior was similar whether the beans were susceptible or resistant to anthracnose. These same Glomerella strains produced thick intracellular hyphae, and eventually acervuli, if host cell death was induced. When Glomerella was co-inoculated with C. lindemuthianum, it readily invaded anthracnose lesions. Thus, the hypothesis was not supported: Glomerella strains from anthracnose lesions do not represent the teleomorphic phase of C. lindemuthianum, and instead appear to be bean epiphytes that opportunistically invade and sporulate in the lesions.

Thermal stability engineering of Glomerella cingulata cutinase.

Cutinase has been ascertained as a biocatalyst for biotechnological and industrial bioprocesses. The Glomerella cingulata cutinase was genetically modified to enhance its enzymatic performance to fulfill industrial requirements. Two sites were selected for mutagenesis with the aim of altering the surface electrostatics as well as removing a potentially deamidation-prone asparagine residue. The N177D cutinase variant was affirmed to be more resilient to temperature increase with a 2.7-fold increase in half-life at 50°C as compared with wild-type enzyme, while, the activity at 25°C is not compromised. Furthermore, the increase in thermal tolerance of this variant is accompanied by an increase in optimal temperature. Another variant, the L172K, however, exhibited higher enzymatic performance towards phenyl ester substrates of longer carbon chain length, yet its thermal stability is inversely affected. In order to restore the thermal stability of L172K, we constructed a L172K/N177D double variant and showed that these two mutations yield an improved variant with enhanced activity towards phenyl ester substrates and enhanced thermal stability. Taken together, our study may provide valuable information for enhancing catalytic performance and thermal stability in future engineering endeavors.

Glomerella truncata: another Glomerella species with an atypical mating system.

In the genus Glomerella all species studied to date do not fit the usual mating system of heterothallic ascomycetes. This study investigated the mating system of G. truncata (anamorph Colletotrichum truncatum), a pathogen responsible for lentil anthracnose. Twenty-two field isolates from the Canadian prairies were crossed in all possible combinations, including selfings. All isolates also were screened for the presence of the MAT1-1 and MAT1-2 idiomorphs by targeting small conserved areas of the MAT genes (the alpha domain and the high mobility group HMG box) with degenerate primers, and a pair of G. truncata-specific HMG primers (CT21HMG) were designed. The results of the classical mating study suggested that G. truncata is heterothallic. Isolates fell into two incompatibility groups, which is consistent with a bipolar mating system but different from what has been described in other Glomerella species. Molecular screening showed that the HMG box used as a marker for the MAT1-2 idiomorph was present in both partners of fertile crosses in G. truncata, unlike in the typical ascomycete system, but as previously described for two other Glomerella species. G. truncata therefore appears to share unusual mating system characteristics with the other Glomerella species studied to date.

Reduction of quinones and phenoxy radicals by extracellular glucose dehydrogenase from Glomerella cingulata suggests a role in plant pathogenicity.

The plant-pathogenic fungus Glomerella cingulata (anamorph Colletotrichum gloeosporoides) secretes high levels of an FAD-dependent glucose dehydrogenase (GDH) when grown on tomato juice-supplemented media. To elucidate its molecular and catalytic properties, GDH was produced in submerged culture. The highest volumetric activity was obtained in shaking flasks after 6 days of cultivation (3400 U l⁻¹, 4.2 % of total extracellular protein). GDH is a monomeric protein with an isoelectric point of 5.6. The molecular masses of the glycoforms ranged from 95 to 135 kDa, but after deglycosylation, a single 68 kDa band was obtained. The absorption spectrum is typical for an FAD-containing enzyme with maxima at 370 and 458 nm and the cofactor is non-covalently bound. The preferred substrates are glucose and xylose. Suitable electron acceptors are quinones, phenoxy radicals, 2,6-dichloroindophenol, ferricyanide and ferrocenium hexafluorophosphate. In contrast, oxygen turnover is very low. The GDH-encoding gene was cloned and phylogenetic analysis of the translated protein reveals its affiliation to the GMC family of oxidoreductases. The proposed function of this quinone and phenoxy radical reducing enzyme is to neutralize the action of plant laccase, phenoloxidase or peroxidase activities, which are increased in infected plants to evade fungal attack.

A Glomerella species phylogenetically related to Colletotrichum acutatum on Norway maple in Massachusetts.

A fungus isolated from Norway maple (Acer platanoides) in the Boston, Massachusetts, area was determined to be a species of Glomerella, the teleomorph of Colletotrin chum acutatum. Pure cultures of the fungus were obtained from discharged ascospores from perithecia in leaf tissue. This fungus was determined to be homothallic based on the observation of perithecial development in cultures of single-spore isolates grown on minimal salts media and with sterile toothpicks. A morphological and molecular analysis was conducted to determine the taxonomic position of this fungus. Parsimony analyses of a combined nucleotide dataset of the ITS and LSU rDNA region, and of the D1-D2 LSU rDNA region, indicated that this species has phylogenetic affinities with Colletotrichum acutatum, C. acutatum f. sp. pineum, C. lupini, C. phormii and G. miyabeana. These results are significant because C. acutatum has not been reported on Acer platanoides. In addition the consistent presence of perithecia on leaf tissue and in culture is unusual for Colletotrichum, suggesting that the teleomorphic state is important in the life cycle of this fungus.

Identification of Glomerella cingulata f. sp phaseoli recombinants by RAPD markers.

We examined the capacity of strains of Glomerella cingulata f. sp phaseoli fungus (Colletotrichum lindemuthianum sexual stage) to form recombinants, using random amplified polymorphic DNA (RAPD). Crosses of all possible combinations between strains 40, 42, 20, 21, 22, 23, 24, 25, and 26 were made on Petri dishes using M3 culture medium. The 42 x 21 cross produced the largest number of perithecia and five asci; the respective ascospores were isolated. RAPD analysis was performed on the parents and descendants. The 62 polymorphic RAPD bands obtained were used to assess the genetic similarity using the method of Sorence and Dice and clustering analysis in the form of a dendrogram by the UPGMA method. The RAPD markers allowed identification of recombinants from the cross between strains 42 and 21 of G. cingulata f. sp phaseoli and 40 ascospores presented 63 and 49% genetic similarity with parents 2 (strain 42) and 1 (strain 21), respectively.

GcSTUA, an APSES transcription factor, is required for generation of appressorial turgor pressure and full pathogenicity of Glomerella cingulata.

Glomerella cingulata, which infects a number of different hosts, gains entry to the plant tissue by means of an appressorium. Turgor pressure generated within the appressorium forces a penetration peg through the plant cuticle. A visible lesion forms as the fungus continues to grow within the host. A G. cingulata homolog (GcSTUA) of the genes encoding Asm1, Phd1, Sok2, Efg1, and StuA transcription factors in Magnaporthe grisea and other fungi was cloned and shown to be required for infection of intact apple fruit and penetration of onion epidermal cells. Mobilization of glycogen and triacylglycerol during formation of appressoria by the GcSTUA deletion mutant appeared normal and melanization of the maturing appressoria was also indistinguishable from that of the wild type. However, GcSTUA was essential for the generation of normal turgor pressure within the appressorium. As is the case for its homologs in other fungi, GcSTUA also was required for the formation of aerial hyphae, efficient conidiation, and the formation of perithecia (sexual reproductive structures).

A phylogenetic evaluation of whether endophytes become saprotrophs at host senescence.

Fungal endophytes and saprotrophs generally play an important ecological role within plant tissues and dead plant material. Several reports based solely on morphological observations have postulated that there is an intimate link between endophytes and saprotrophs. This study aims to provide valuable insight as to whether some endophytic fungi manifest themselves as saprotrophs upon host decay. Ribosomal DNA-based sequence comparison and phylogenetic relationships from 99 fungal isolates (endophytes, mycelia sterilia, and saprotrophs) recovered from leaves and twigs of Magnolia liliifera were investigated in this study. Molecular data suggest there are fungal taxa that possibly exist as endophytes and saprotrophs. Isolates of Colletotrichum, Fusarium, Guignardia, and Phomopsis, which are common plant endophytes, have high sequence similarity and are phylogenetically related to their saprotrophic counterparts. This provides evidence to suggest that some endophytic species change their ecological strategies and adopt a saprotrophic lifestyle. The implication of these findings on fungal biodiversity and host specificity is also discussed.

Molecular cloning and heterologous expression of a new xylanase gene from Plectosphaerella cucumerina.

A gene encoding a new xylanase, named xynZG, was cloned by the genome-walking PCR method from the nematophagous fungus Plectosphaerella cucumerina. The genomic DNA sequence of xynZG contains a 780 bp open reading frame separated by two introns with the sizes of 50 and 46 bp. To our knowledge, this would be the first functional gene cloned from P. cucumerina. The 684 bp cDNA was cloned into vector pHBM905B and transformed into Pichia pastoris GS115 to select xylanase-secreting transformants on RBB-xylan containing plate. The optimal secreting time was 3 days at 25 degrees C and enzymatic activities in the culture supernatants reached the maximum level of 362 U ml(-1). The molecular mass of the enzyme was estimated to be 19 kDa on SDS-PAGE. The optimal pH and temperature of the purified enzyme is 6 and 40 degrees C, respectively. The purified enzyme is stable at room temperature for at least 10 h. The Km and Vmax values for birchwood xylan are 2.06 mg ml(-1) and 0.49 mmol min(-1)mg(-1), respectively. The inhibitory effects of various mental ions were investigated. It is interesting to note that Cu2+ ion, which strongly inhibits most other xylanases studied, reduces enzyme activity by only 40%. Furthermore, enzyme activity is unaffected by EDTA even at a concentration of 5 mM.

Heterothallic mating observed between Mexican isolates of Glomerella lindemuthiana.

Although several reports have described the occurrence of the teleomorphic state of Glomerella lindemuthiana (anamorph, Colletotrichum lindemuthianum), there has been a lack of continuity in this research. To identify G. lindemuthiana isolates capable of developing the teleomorphic state, 19 Mexican isolates were analyzed. Three types of response were observed: (i) negative, where only mycelial growth with or without acervuli was observed; (ii) potential, where in addition to the above, spherical perithecia-like structures were observed; (iii) positive, where perithecia containing asci and ascospores were observed. All strains were self-sterile and only one combination of strains produced fertile perithecia. From this fertile combination 168 individual ascospore cultures were isolated, including five from a single ascus. Forty-four monoascospore cultures were characterized with AFLP, confirming that these individuals were progeny from a sexual cross between the original two G. lindemuthiana isolates and that sexual reproduction in G. lindemuthiana is heterothallic in nature. Analysis of the parental strains with degenerate PCR primers indicated that sequences homologous to the HMG box of the MAT1-2 idiomorph are present in both parental isolates. This supports previous observations in other Glomerella species where the standard ascomycete configuration of distinct idiomorphs at the MAT locus does not hold true. The significance of these results is discussed.

Unprecedented mechanism of chain length determination in fungal aromatic polyketide synthases.

Fungal aromatic polyketides show remarkable structural diversity fundamentally derived from variations in chain length and cyclization pattern. Their basic skeletons are synthesized by multifunctional iterative type I polyketide synthases (PKSs). Recently, we have found that the C-terminal thioesterase (TE)-like domain of Aspergillus nidulans WA catalyzes Claisen-type cyclization to form the B-ring of naphthopyrone YWA1. Here we report the unprecedented mechanism of chain length determination by the C-terminal TE-like domain of Colletotrichum lagenarium PKS1, which, in addition to catalyzing Claisen-type cyclization, intercepts the polyketomethylene intermediate from the acyl carrier protein domain during the condensation reaction to produce shorter chain length products. This chain length determination system is novel among PKSs, including bacterial and plant PKSs. The functional diversity of the TE-like domain directly influences the structural diversity of aromatic polyketides in C. lagenarium PKS1.

Engineering a genetic transformation system for Colletotrichum acutatum, the causal fungus of lime anthracnose and postbloom fruit drop of citrus.

Postbloom fruit drop (PFD) of citrus is caused by Colletotrichum acutatum. PFD isolates infect flower petals, induce abscission of small fruit and can cause severe yield loss on most citrus cultivars. Isolates from Key lime anthracnose (KLA) cause that disease on the Mexican lime, but also cause PFD on sweet orange. Both PFD and KLA isolates exhibited resistance to the common selection agents including hygromycin, bialaphos, benomyl and geneticin/G418. A genetic transformation system was developed for C. acutatum to confer resistance to sulfonylurea (chlorimuron ethyl) by expressing an acetolactate synthase gene (sur) cassette from Magnaporthe grisea. The protocol was tested on 11 different KLA and PFD isolates. The transformation frequencies were highly variable among isolates and among experiments (0-17.9 per microg circular DNA using 10(7) protoplasts). Southern blot analysis of transformants indicated that the plasmid vector was randomly integrated in multiple copies into the genome of C. acutatum. Addition of restriction enzymes or use of a vector with homologous sequences did not change the transformation frequencies, but tended to reduce the number integrated. Over 97% of the transformants retained the sulfonylurea resistance phenotype under non-selective conditions. Of 300 transformants tested, three were unable to cause necrotic lesions on detached Key lime leaves. The transformation method opens up opportunities for the genetic manipulation of C. acutatum.

Mating system of the filamentous ascomycete, Glomerella cingulata.

Mating in heterothallic filamentous ascomycetes is typically controlled by a single mating-type locus with two alternate alleles or idiomorphs. In this study, five self-sterile strains of Glomerella cingulata from pecan were crossed in all possible combinations. Four of the five strains could be placed into two mating-type groups, but the fifth strain was sexually compatible with all of the other strains. Single ascospore progeny were isolated from each of the successful crosses, tested for self-fertility, and backcrossed with both parents. In addition, subsets of F1 isolates were crossed with all five of the original strains from pecan and in all possible combinations with each other. Results from the crosses showed that the ascospore progeny had stably inherited the mating pattern of one of the parental strains and that the mating type had segregated 1:1 among the F1 isolates. Furthermore, the five strains from pecan were sexually compatible with five additional heterothallic strains in all but one combination. Data from these experiments are consistent with a mating system composed of a single mating-type locus with multiple alternate alleles. We believe that this is the first report of this type of mating system for an ascomycete species.