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 apple. 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, the common pathogen of GLS and ABR, and to evaluate the genes involvement on pathogenicity.Results: A relatively large difference was discovered between the GLS- and ABR-isolate, and quite a number of 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. A number of genes associate 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 secretion of extracellular enzymes and melanin accumulation were up-regulated. Four genes associated with degradation of host cell wall, three genes involved in degradation of cellulose, and one gene involved in degradation of xylan were revealed and all up-regulated. In addition, genes involved in melanin synthesis, such as tyrosinase and glucosyltransferase, were highly up-regulated.Conclusions: The penetration ability, pathogenicity of GLS-isolate was greater than that ABR-isolate, which might be indicate that GLS-isolate originated from ABR-isolates by mutation. These results contributed to highlight the importance to investigate such DEGs between GLS- and ABR-isolate in depth.

First Report of Colletotrichum aenigma Causing Apple Glomerella Leaf Spot on the Granny Smith Cultivar in China

Glomerella leaf spot (GLS) is a devastating fungal disease causing pre-mature defoliation on apple (Malus domestica). It was first reported in 1970s and since then has been reported in North America, South America and Asia. GLS disease is caused by Colletotrichum fungi and the pathogens are genetically diverse, encompassing at least nine species belonging to three species complexes (Velho et al. 2018). In August 2018, disease with sudden leaf necrosis symptom, typical of GLS symptom appearance, occurred in a Granny Smith orchard in Wugong county, China, over 70% tree leaves bared brown and necrotic lesions. Small leaf tissues (3-4 mm2) cut from lesion margins were surface sterilized for 30 s in 3% NaClO and 30 s in 75% ethanol, followed by rinsing three times in sterile water before transferring onto potato dextrose agar (PDA) plates (25 ± 2°C). Seven isolates were obtained, all producing round cottony colonies on PDA, being white to pale on the upper side and dark green on the reverse side. Conidia were single-celled, cylindrical and transparent (17.33 ± 1.29 × 5.11 ± 0.77 μm, n=50). Appressoria were single-celled, thick-walled, dark brown, oval or irregular shaped, sometimes lobed (9.07 ± 0.88 × 6.66 ± 0.33 μm, n=50). The morphological and cultural characteristics of the fungal isolates matched the descriptions of Colletotrichum aenigma (Weir et al. 2012). To confirm the species identity, genomic DNAs were isolated from two representative isolates (QSG1 and QSY1), and the internal transcribed spacer (ITS), actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-tubulin (TUB2), manganese-superoxide dismutase (SOD2), chitin synthase (CHS), and calmodulin (CAL) regions were amplified by PCR using reported primers (Weir et al. 2012). The sequences were deposited in GenBank (Accession Nos. MT872061, MT873580, MT873581, MT888183, MT888185, MT888187 and MT888189 for QSG1, and MT834933, MT835166, MT873579, MT888182, MT888184, MT888186 and MT888188 for QSY1). BLASTn search against GenBank nr database showed that ITS sequences of the two strains showed high nucleotide identity (over 99%) to sequences derived from the Colletotrichum gloeosporioides species complex (CGSC). Further concatenated phylogenetic analysis with reported CGSC strains (Weir et al. 2012) placed QSG1 and QSY1 in the clade of C. aenigma. To fulfill Koch’s postulates, field pathogenicity test was performed. The experiment was performed in an orchard located in Yangling in September 2020, with the daily average temperature ranging between 15 - 20 ℃. Healthy ‘Granny Smith’ leaves were surface sterilized with 70% alcohol and inoculated with conidial suspension (106 conidia/mL) using cotton swabs. For each isolate, 10 leaf inoculations were performed. Inoculation with distilled water served as a negative control. Inoculated leaves were covered with plastic bags to maintain high humidity and the bags were removed at 48 hours post inoculation (hpi). Conidium-inoculated leaves started to exhibit GLS-resembling necrotic lesions from 5 dpi onward. The lesion extent, however, varied among inoculated leaves, ranging from blurry, small-sized lesions to blight of entire leaf. In contrast with conidium inoculations, water-inoculated leaves remained asymptomatic until 14 dpi. Re-isolated fungi from the symptomic leaf tissues were identical to C. aenigma in morphological appearance. Taken together, this is the first report of GLS on ‘Granny Smith’ apple, and the first report of C. aenigma causing apple GLS in China. This information should provide important guideline for developing field control practices of GLS.

Transcription Factor CfSte12 of Colletotrichum fructicola Is a Key Regulator of Early Apple Glomerella Leaf Spot Pathogenesis

ABSTRACT Glomerella leaf spot (GLS), caused by Colletotrichum fructicola, is a rapidly emerging disease leading to defoliation, fruit spot, and storage fruit rot on apple in China. Little is known about the mechanisms of GLS pathogenesis. Early transcriptome analysis revealed that expression of the zinc finger transcription factor Ste12 gene in C. fructicola (CfSte12) was upregulated in appressoria and leaf infection. To investigate functions of CfSte12 during pathogenesis, we constructed gene deletion mutants (ΔCfSte12) by homologous recombination. Phenotypic analysis revealed that CfSte12 was involved in pathogenesis of nonwounded apple fruit and leaf, as well as wounded apple fruit. Subsequent histological studies revealed that loss of pathogenicity by ΔCfSte12 on apple leaf was expressed as defects of conidium germination, appressorium development, and appressorium-mediated penetration. Further RNA sequencing-based transcriptome comparison revealed that CfSte12 modulates the expression of genes related to appressorium function (e.g., genes for the tetraspanin PLS1, Gas1-like proteins, cutinases, and melanin biosynthesis) and candidate effectors likely involved in plant interaction. In sum, our results demonstrated that CfSte12 is a key regulator of early apple GLS pathogenesis in C. fructicola. In addition, CfSte12 is also needed for sexual development of perithecia and ascospores. IMPORTANCE Glomerella leaf spot (GLS) is an emerging fungal disease of apple that causes huge economic losses in Asia, North America, and South America. The damage inflicted by GLS manifests in rapid necrosis of leaves, severe defoliation, and necrotic spot on the fruit surface. However, few studies have addressed mechanisms of GLS pathogenesis. In this study, we identified and characterized a key pathogenicity-related transcription factor, CfSte12, of Colletotrichum fructicola that contributes to GLS pathogenesis. We provide evidence that the CfSte12 protein regulates many important pathogenic processes of GLS, including conidium germination, appressorium formation, appressorium-mediated penetration, and colonization. CfSte12 also impacts development of structures needed for sexual reproduction which are vital for the GLS disease cycle. These results reveal a key pathogenicity-related transcription factor, CfSte12, in C. fructicola that causes GLS.

SCS427 Elenise: late-ripening apple variety of good storability and resistance to Glomerella Leaf Spot

‘SCS427 Elenise’ (parents: Imperatriz and Cripps Pink) was released for being an apple variety resistant to Glomerella Leaf Spot, for producing red apple with late maturation, with high conservation capacity and pulp texture equivalent to the fruit Cripps Pink’s parent, but with less acidic flavor. It represents the opportunity to extend the harvest window and offer the market high quality apples throughout the inter-harvest period.