Rapid Identification of Phytotoxins Produced by Glomerella cingulata Using High-Resolution Mass Spectrometry-Based Qualification, Targeted Structural Confirmation and Their Characteristics Investigation.

Glomerella cingulata is a pathogenic fungus that can cause apple Glomerella leaf spot (GLS), a new and destructive apple disease in China. Phytotoxins are important factors closely related to the disease process, but there is still no report on the phytotoxins of G. cingulata. The aim of this study was to rapidly identify the phytotoxins of this pathogen using a strategy of HRMS-based preliminary qualification, followed by targeted structure confirmation and also investigation of phytotoxicity characteristics. First, the crude toxin sample was directly analyzed by the UPLC-HRMS and GC-MS, and the data were processed to screen for possible phytotoxic compounds using MS library and the phytotoxicity-related literature. The reference standards of credible phytotoxic compounds were then subjected to targeted structure validation (signal comparison between standards and compounds in crude toxin via HPLC-DAD, UPLC-MS/MS, and GC-MS), and also the phytotoxicity assay. The results confirmed six phytotoxins produced by G. cingulata, namely 5-hydroxymethyl-2-furancarboxylic acid (HMFCA), 2,5-bis(hydroxymethyl)furan (BHMF), 2-furoic acid (FA), 2,3-butanediol, trans-aconitic acid (TAA), and cis-aconitic acid (CAA). Of these, HMFCA and TAA exhibited greater phytotoxicity. Main characteristics: All of them were non-host-selective toxins, and toxins were synergistically phytotoxic to the host when mixed. BHMF, HMFCA, FA, TAA, and CAA could be commonly produced by all tested strains, and their phytotoxicity can be significantly inhibited or even eliminated at high temperatures or high pH. The elucidation of the phytotoxins of G. cingulata in this work could provide information on the pathogenesis and control of apple GLS.

Colletotrichum species causing Glomerella leaf spot and apple bitter rot in the southeastern United States exhibit disparities in relative frequency, morphological phenotype, and QoI sensitivity.

Glomerella leaf spot (GLS), Glomerella fruit rot (GFR) and apple bitter rot (ABR), caused by Colletotrichum spp. are amongst the most devastating apple diseases in the southeastern United States. While several species have been identified as causal pathogens of GLS, GFR, and ABR, their relative frequency and fungicide sensitivity status in the southeastern U.S. is unknown. In total, 381 Colletotrichum isolates were obtained from symptomatic leaves and fruit from 18 conventionally managed apple orchards and two baseline populations in western North Carolina and Georgia in 2016 and 2017. Multilocus DNA sequence analysis revealed that C. chrysophilum was the predominant cause of GLS and GFR, and C. fioriniae was the causal agent of ABR. Baseline and commercial populations of Colletotrichum spp. were evaluated for sensitivity to pyraclostrobin and trifloxystrobin and no statistical differences in sensitivity between the two species were observed for conidial germination. However, EC50 values were significantly lower for C. fioriniae compared to C. chrysophilum for both fungicides regarding mycelial inhibition. Isolates recovered from commercial orchards revealed that 5 populations of C. chrysophilum and 1 population of C. fioriniae had reduced sensitivity to trifloxystrobin, and 1 C. fioriniae population had reduced sensitivity to pyraclostrobin via conidial germination assays. The cytb gene for 27 isolates of C. fioriniae, C. chrysophilum, and C. fructicola with different QoI sensitivities revealed the G143A mutation in a single isolate of C. chrysophilum with insensitivity to both fungicides. Results of these studies suggest that two Colletotrichum spp. predominantly cause GLS and ABR in the southeastern U.S. and that a reduction in sensitivity to some QoI fungicides may be responsible for control failures. This study also provides basis for monitoring shifts in QoI sensitivity in Colletotrichum spp. causing disease on apple in the southeastern U.S.

Colletotrichum Species Associated with Apple Bitter Rot and Glomerella Leaf Spot: A Comprehensive Overview.

Species of the genus Colletotrichum are among the most important plant pathogens globally, as they are capable of infecting many hosts-apple (Malus spp.) and other fruit and woody plant species-but also vegetable crops, cereals, legumes, and other annual and perennial herbaceous plants. The apple (Malus spp.) is attacked by various species from the genus Colletotrichum, whereby 27 different species from this genus have been described as the causative agents of apple bitter rot (ABR) and 15 as the cause of Glomerella leaf spot (GLS). These species generally belong to one of three species complexes: Colletotrichum acutatum, Colletotrichum gloeosporioides, and Colletotrichum boninense. The largest number of apple pathogens of the genus Colletotrichum belong to the species complex C. acutatum and C. gloeosporioides. However, further data on these species and the interactions between the species complexes of the genus Colletotrichum that cause these two apple diseases is needed for the development of effective control measures, thus ensuring successful and profitable apple cultivation. To contribute to this endeavor, a comprehensive review of the causative agents of ABR and GLS from the genus Colletotrichum is provided. In addition to presenting the species' current names, distribution, economic significance, and the symptoms they cause in apple, their development cycle, epidemiology, and molecular detection strategies are described, with a particular emphasis on control measures.

The HD-Zip I transcription factor MdHB-7 negatively regulates resistance to Glomerella leaf spot in apple.

Glomerella leaf spot (GLS), caused by Colletotrichum fructicola (Cf), has been one of the main fungal diseases afflicting apple-producing areas across the world for many years, and it has led to substantial reductions in apple output and quality. HD-Zip transcription factors have been identified in several species, and they are involved in the immune response of plants to various types of biotic stress. In this study, inoculation of MdHB-7 overexpressing (MdHB-7-OE) and interference (MdHB-7-RNAi) transgenic plants with Cf revealed that MdHB-7, which encodes an HD-Zip transcription factor, adversely affects GLS resistance. The SA content and the expression of SA pathway-related genes were lower in MdHB-7-OE plants than in 'GL-3' plants; the content of ABA and the expression of ABA biosynthesis genes were higher in MdHB-7-OE plants than in 'GL-3' plants. Further analysis indicated that the content of phenolics and chitinase and ß-1, 3 glucanase activities were lower and H2O2 accumulation was higher in MdHB-7-OE plants than in 'GL-3' plants. The opposite patterns were observed in MdHB-7-RNAi apple plants. Overall, our results indicate that MdHB-7 plays a negative role in regulating defense against GLS in apple, which is likely achieved by altering the content of SA, ABA, polyphenols, the activities of defense-related enzymes, and the content of H2O2.

MdWRKY71 promotes the susceptibility of apple to Glomerella leaf spot by controlling salicylic acid degradation.

Glomerella leaf spot (GLS), a fungal disease caused by Colletotrichum fructicola, severely affects apple (Malus domestica) quality and yield. In this study, we found that the transcription factor MdWRKY71 was significantly induced by C. fructicola infection in the GLS-susceptible apple cultivar Royal Gala. The overexpression of MdWRKY71 in apple leaves resulted in increased susceptibility to C. fructicola, whereas RNA interference of MdWRKY71 in leaves showed the opposite phenotypes. These findings suggest that MdWRKY71 functions as a susceptibility factor for the apple-C. fructicola interaction. Furthermore, MdWRKY71 directly bound to the promoter of the salicylic acid (SA) degradation gene Downy Mildew Resistant 6 (DMR6)-Like Oxygenase 1 (DLO1) and promoted its expression, resulting in a reduced SA level. The sensitivity of 35S:MdWRKY71 leaves to C. fructicola can be effectively alleviated by knocking down MdDLO1 expression, confirming the critical role of MdWRKY71-mediated SA degradation via regulating MdDLO1 expression in GLS susceptibility. In summary, we identified a GLS susceptibility factor, MdWRKY71, that targets the apple SA degradation pathway to promote fungal infection.

The MdVQ37-MdWRKY100 complex regulates salicylic acid content and MdRPM1 expression to modulate resistance to Glomerella leaf spot in apples.

Glomerella leaf spot (GLS), caused by the fungus Colletotrichum fructicola, is considered one of the most destructive diseases affecting apples. The VQ-WRKY complex plays a crucial role in the response of plants to biotic stresses. However, our understanding of the defensive role of the VQ-WRKY complex on woody plants, particularly apples, under biotic stress, remains limited. In this study, we elucidated the molecular mechanisms underlying the defensive role of the apple MdVQ37-MdWRKY100 module in response to GLS infection. The overexpression of MdWRKY100 enhanced resistance to C. fructicola, whereas MdWRKY100 RNA interference in apple plants reduced resistance to C. fructicola by affecting salicylic acid (SA) content and the expression level of the CC-NBS-LRR resistance gene MdRPM1. DAP-seq, Y1H, EMSA, and RT-qPCR assays indicated that MdWRKY100 inhibited the expression of MdWRKY17, a positive regulatory factor gene of SA degradation, upregulated the expression of MdPAL1, a key enzyme gene of SA biosynthesis, and promoted MdRPM1 expression by directly binding to their promotors. Transient overexpression and silencing experiments showed that MdPAL1 and MdRPM1 positively regulated GLS resistance in apples. Furthermore, the overexpression of MdVQ37 increased the susceptibility to C. fructicola by reducing the SA content and expression level of MdRPM1. Additionally, MdVQ37 interacted with MdWRKY100, which repressed the transcriptional activity of MdWRKY100. In summary, these results revealed the molecular mechanism through which the apple MdVQ37-MdWRKY100 module responds to GLS infection by regulating SA content and MdRPM1 expression, providing novel insights into the involvement of the VQ-WRKY complex in plant pathogen defence responses.

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.

GDSL Esterase/Lipase GELP1 Involved in the Defense of Apple Leaves against Colletotrichum gloeosporioides Infection.

GDSL esterases/lipases are a subclass of lipolytic enzymes that play critical roles in plant growth and development, stress response, and pathogen defense. However, the GDSL esterase/lipase genes involved in the pathogen response of apple remain to be identified and characterized. Thus, in this study, we aimed to analyze the phenotypic difference between the resistant variety, Fuji, and susceptible variety, Gala, during infection with C. gloeosporioides, screen for anti-disease-associated proteins in Fuji leaves, and elucidate the underlying mechanisms. The results showed that GDSL esterase/lipase protein GELP1 contributed to C. gloeosporioides infection defense in apple. During C. gloeosporioides infection, GELP1 expression was significantly upregulated in Fuji. Fuji leaves exhibited a highly resistant phenotype compared with Gala leaves. The formation of infection hyphae of C. gloeosporioides was inhibited in Fuji. Moreover, recombinant His:GELP1 protein suppressed hyphal formation during infection in vitro. Transient expression in Nicotiana benthamiana showed that GELP1-eGFP localized to the endoplasmic reticulum and chloroplasts. GELP1 overexpression in GL-3 plants increased resistance to C. gloeosporioides. MdWRKY15 expression was upregulated in the transgenic lines. Notably, GELP1 transcript levels were elevated in GL-3 after salicylic acid treatment. These results suggest that GELP1 increases apple resistance to C. gloeosporioides by indirectly regulating salicylic acid biosynthesis.

γ-Aminobutyric acid plays a key role in alleviating Glomerella leaf spot in apples.

The fungal disease Glomerella leaf spot (GLS) seriously impacts apple production. As a nonprotein amino acid, γ-aminobutyric acid (GABA) is widely involved in biotic and abiotic stresses. However, it is not clear whether GABA is involved in a plant's response to GLS, nor is its molecular mechanism understood. Here, we found that exogenous GABA could significantly alleviate GLS, reduce lesion lengths, and increase antioxidant capacity. MdGAD1 was identified as a possible key gene for GABA synthesis in apple. Further analysis indicated that MdGAD1 promoted antioxidant capacity to improve apple GLS resistance in transgenic apple calli and leaves. Yeast one-hybrid analysis identified the transcription factor MdWRKY33 upstream of MdGAD1. Electrophoretic mobility shift assay, ß-glucuronidase activity, and luciferase activity further supported that MdWRKY33 bound directly to the promoter of MdGAD1. The content of GABA and the transcription level of MdGAD1 in the MdWRKY33 transgenic calli were higher than that of the wild type. When MdWRKY33 transgenic calli and leaves were inoculated with GLS, MdWKRY33 positively regulated resistance to GLS. These results explained the positive regulatory effects of GABA on apple GLS and provided insight into the metabolic regulatory network of GABA.

Detection of Cytb Point Mutation (G143A) that Confers High-Level Resistance to Pyraclostrobin in Glomerella cingulata Using LAMP Method.

Glomerella leaf spot (GLS) caused by Glomerella cingulata is a newly emerging disease that results in severe defoliation and fruit spots in apples. In China, the compound of pyraclostrobin and tebuconazole was registered to control GLS in 2018 and has achieved excellent control efficiency. In this study, we showed that the high-level resistant isolates of G. cingulata to pyraclostrobin, caused by the point mutation at codon 143 (GGT→GCT, G143A) in the cytochrome b gene, has appeared in apple orchards in Shandong Province in 2020, and the resistance frequency was 4.8%. Based on the genotype of the resistant isolates, we developed a loop-mediated isothermal amplification (LAMP) assay for detection of the pyraclostrobin resistance. The LAMP assay was demonstrated to have good specificity, sensitivity, and repeatability, and it exhibited high accuracy in detecting pyraclostrobin resistance in the field. This study reported the resistance status of GLS to pyraclostrobin in Shandong Province and developed a molecular tool for the detection of pyraclostrobin resistance, which is of practical significance for the scientific control of GLS.

Identification and Fungicide Sensitivity of Colletotrichum spp. from Apple Flowers and Fruitlets in Brazil.

Glomerella leaf spot (GLS) and bitter rot (BR), caused by Colletotrichum spp., are major diseases on apple in southern Brazil. Among integrated pest management tools for disease management in commercial orchards, fungicides remain an important component. This study aimed to identify Colletotrichum spp. from cultivar Eva in Paraná state orchards; evaluate their in vitro sensitivity to cyprodinil, tebuconazole, iprodione, and fluazinam; and determine the baseline in vitro sensitivity of these isolates to benzovindiflupyr and natamycin. Most isolates belonged to Colletotrichum melonis and C. nymphaeae of the C. acutatum species complex. The two species varied in sensitivity to fluazinam and tebuconazole, but no variability was found for any other fungicide. The lowest 50% effective concentration (EC50) values of Colletotrichum spp. were observed for cyprodinil (mean EC50 < 0.02) and benzovindiflupyr (mean EC50 < 0.05); EC50 values were intermediate for fluazinam (mean EC50 < 0.33) and tebuconazole (mean EC50 < 0.14), and they were highest for natamycin (mean EC50 < 5.56) and iprodione (mean EC50 > 12). Cyprodinil and fluazinam are registered for use in Brazil for apple disease management but not specifically for GLS and BR. Tebuconazole is one of the few products registered for Colletotrichum spp. control in apples. In conclusion, flowers and fruitlets can serve as sources of inoculum for GLS and BR disease; C. acutatum was the predominant species complex in these tissues; cyprodinil and fluazinam applications may suppress GLS and BR; and benzovindiflupyr and natamycin warrant further investigation for GLS and BR disease control of apple due to comparably high in vitro sensitivity.

First report of Preharvest Decay caused by Colletotrichum chrysophilum on apples in Italy (South Tyrol).

Apple (Malus × domestica Borkh.) is economically the most important fruit crop in South Tyrol (Italy). At the end of the growing season 2020, necrotic lesions and chlorosis developed on leaves and premature leaf dropping was observed on the cultivars Gala, Granny Smith and Cripps Pink(cov)/Rosy Glow(cov) in the Etsch/Adige valley. After the appearance of these symptoms, small circular brownish spots were observed on above 90 % of apples in the respective orchards. Fungal isolates were obtained from symptomatic apples by culturing small portions of fruit flesh from the lesion margin on potato dextrose agar (PDA) at 25°C in the dark. The colonies showed a white to peachy color on the upper surface and were greyish on the reverse side. Conidia were cylindrical, predominantly rounded and averaged 16.39 ± 1.37 µm and 5.75 ± 0.81 µm (n = 33), consistent morphological characteristics as described recently in Fuentes-Aragón et al. (2021). A multi-locus sequence analysis according to Astolfi et al. (2022) and Weir et al. (2022) was conducted based on the internal transcribed spacer (ITS) region and on fragments of actin (ACT), DNA-(apurinic or apyrimidinic site) lyase (APN2), calmodulin (CAL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutamine synthetase (GS), beta-tubulin (TUB2), and intergenic spacer and partial mating type (Mat1-2) genes. MegaBlast analysis revealed 100 % identity to the reference genome of C. chrysophilum for ITS (NR_160821), for ACT (KX093982), APN2 (KX094018), CAL (KX094063), GAPDH (KX094183), 99.29 % for TUB2 (KX094285), 99.85 % for Mat1-2 (KX094325) and 99.87 % for GS (KX094204). Sequences were deposited in GenBank (ITS: OK485032; ACT: OK539650; APN2: ON624100; CAL: ON624104; GAPDH: OK539654; GS: ON624108; TUB2: OK539658; Mat1-2: ON645973). Pathogenicity assays were performed on wounded and unwounded apples of cvs. Gala and Rosy Glow(cov). Apples of both cvs. were wounded with a sterile needle. For each variant, 8 fruits were inoculated with 10 µL spore suspension (1 × 106 spores mL-1) in 0.05 % Tween®20 or with 10 µL 0.05 % Tween®20 as control. The apples were put into plastic boxes containing moist tissue and incubated at 25°C, 100 % relative humidity and 12 h photoperiod. First lesions appeared on inoculated wounded fruits after 5 days, whereas unwounded and control fruits remained asymptomatic. After 15 days, symptoms could be observed only on inoculated wounded apples with spore suspension: on 95 % of individual wounds of Rosy Glow(cov) and on 77.5 % of Gala. Koch's postulates were fulfilled by re-isolating the fungus and by identifying the re-isolates as C. chrysophilum (ITS: OK485033-OK485035; ACT: OK539651-OK539653; APN2: ON624101-ON624103; CAL: ON624105-ON624107; GAPDH: OK539655-OK539657; GS: ON624109; TUB2: OK539659-OK539661; Mat1-2: ON645974-ON645976). To date, only one study confirmed C. chrysophilum as causal agent of apple bitter rot in Europe (Cabrefiga et al. 2022). Recently, Astolfi et al. (2022) reclassified C. chrysophilum as the main causal agent of GLS on apples in Southern Brazil and Uruguay. The authors stressed that C. chrysophilum might also be the potential agent of GLS in Europe (Astolfi et al. 2022). This confirms the observations made in 2020 in South Tyrol, where massive leaf spots preceded the symptoms on fruit. To the best of our knowledge, this is the first report of a preharvest decay on apples caused by C. chrysophilum in Italy (South Tyrol).

Diversity of Colletotrichum Species Causing Apple Bitter Rot and Glomerella Leaf Spot in China.

Bitter rot and Glomerella leaf spot (GLS) of apples, caused by Colletotrichum species, are major diseases of apples around the world. A total of 98 isolates were obtained from apple fruits with bitter rot, and 53 isolates were obtained from leaves with leaf spot in the primary apple production regions in China. These isolates were characterized morphologically, and five gene regions (ITS, ACT, GAPDH, CHS-1 and TUB2) were sequenced for each isolate. A phylogenetic analysis, combined with a comparison of the morphological, cultural and pathogenic characters, sorted bitter rot isolates into six species: C. alienum, C. fructicola, C. gloeosporioides sensu stricto, C. nymphaeae, C. siamense and one new species, C. orientalis Dandan Fu & G.Y. Sun. Among these, C. siamense was the predominant pathogen associated with bitter rot. Isolates from leaf spot were identified as two species, C. aenigma and C. fructicola. This is the first report of C. orientalis as an apple bitter rot pathogen worldwide, and the results provide important insights into the diversity of Colletotrichum species in China.

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.

Pest categorisation of Colletotrichum fructicola.

The EFSA Plant Health Panel performed a pest categorisation of Colletotrichum fructicola Prihast., a well-defined polyphagous fungus of the C. gloeosporioides complex which has been reported from all the five continents to cause anthracnose, bitter rot and leaf spotting diseases on over 90 cultivated and non-cultivated woody or herbaceous plant species. The pathogen is not included in EU Commission Implementing Regulation 2019/2072. Because of the very wide host range, this pest categorisation focused on Camellia sinensis, Citrus sinensis, C. reticulata, Fragaria × ananassa, Malus domestica, M. pumila, Persea americana, Prunus persica, Pyrus pyrifolia and P. bretschneideri for which there was robust evidence that C. fructicola was formally identified by morphology and multilocus gene sequencing analysis. Host plants for planting and fresh fruits are the main pathways for the entry of the pathogen into the EU. There are no reports of interceptions of C. fructicola in the EU. The pathogen has been reported from Italy and France. The host availability and climate suitability factors occurring in some parts of the EU are favourable for the establishment of the pathogen. Economic impact on the production of the main hosts is expected if establishment occurs. Phytosanitary measures are available to prevent the re-introduction of the pathogen into the EU. Although the pathogen is present in the EU, there is a high uncertainty on its actual distribution in the territory because of the re-evaluation of Colletotrichum taxonomy and the lack of systematic surveys. Therefore, the Panel cannot conclude with certainty on whether C. fructicola satisfies the criterium of being present but not widely distributed in the EU to be regarded as a potential Union quarantine pest unless systematic surveys for C. fructicola are conducted and Colletotrichum isolates from the EU in culture collections are re-evaluated.

Carbamoyl Phosphate Synthase Subunit CgCPS1 Is Necessary for Virulence and to Regulate Stress Tolerance in Colletotrichum gloeosporioides.

Glomerella leaf spot (GLS) is a severe infectious disease of apple whose infective area is growing gradually and thus poses a huge economic threat to the world. Different species of Colletotrichum including Colletotrichum gloeosporioides are responsible for GLS. For efficient GLS control, it is important to understand the mechanism by which the cruciferous crops and C. gloeosporioides interact. Arginine is among one of the several types of amino acids, which plays crucial role in biochemical and physiological functions of fungi. The arginine biosynthesis pathway involved in virulence among plant pathogenic fungi is poorly understood. In this study, CgCPS1 gene encoding carbamoyl phosphate synthase involved in arginine biosynthesis has been identified and inactivated experimentally. To assess the effects of CgCPS1, we knocked out CgCPS1 in C. gloeosporioides and evaluated its effects on virulence and stress tolerance. The results showed that deletion of CgCPS1 resulted in loss of pathogenicity. The Δcgcps1 mutants showed slow growth rate, defects in appressorium formation and failed to develop lesions on apple leaves and fruits leading to loss of virulence while complementation strain (CgCPS1-C) fully restored its pathogenicity. Furthermore, mutant strains showed extreme sensitivity to high osmotic stress displaying that CgCPS1 plays a vital role in stress response. These findings suggest that CgCPS1 is major factor that mediates pathogenicity in C. gloeosporioides by encoding carbamoyl phosphate that is involved in arginine biosynthesis and conferring virulence in C. gloeosporioides.

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

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.

Extracellular enzymes of Colletotrichum fructicola isolates associated to Apple bitter rot and Glomerella leaf spot.

Colletotrichum fructicola causes two important diseases on apple in Southern Brazil, bitter rot (ABR) and Glomerella leaf spot (GLS). In this pathosystem, the Colletotrichum ability to cause different symptoms could be related to differences of extracellular enzymes produced by the fungi. Thus, the objectives of this study were to compare the production of these enzymes between ABR- and GLS-isolate in vitro and to evaluate their involvement on infected apple leaves with C. fructicola. In agar plate enzymatic assay, ABR- showed significantly higher amylolytic and pectolytic activity than GLS-isolate. In contrast, for lipolytic and proteolytic no significant differences were observed between isolates. In culture broth, ABR-isolate also had higher activity of pectin lyase (PNL), polygalacturonase (PG) and laccase (LAC). Notably, LAC was significantly five-fold higher in ABR- than GLS-isolate. On the other hand, in infected apple leaves no significant difference was observed between isolates for PNL, PG and LAC. Although differences in extracellular enzymes of ABR- and GLS-isolate have not been observed in vivo, these results contributed to highlight the importance to investigate such enzymes in depth.

Modulation of oxidative responses by a virulent isolate of Colletotrichum fructicola in apple leaves.

Apple bitter rot (ABR) and Glomerella leaf spot (GLS) can be caused by Colletotrichum fructicola. Although both diseases can occur simultaneously in orchards, some isolates show clear organ specialization. Thus, this work was aimed to compare microscopically the development of preinfective structures of ABR- and GLS isolates and their impact on the enzymatic oxidant defense system during the leaf infection process. On leaves, conidial germlings of GLS-isolate formed appressoria mostly sessile. In contrast, those of ABR-isolate were pedicellate and formed multiple melanized appressoria probably as a sign of unsuccessful infection attempts. Neither ABR- nor GLS isolate triggered hypersensitive response in apple leaves. In overall, the activity of scavenging enzymes was higher and long-lasting in leaves inoculated by GLS- than by ABR isolate and control. Guaiacol peroxidase, catalase, and glutathione reductase had activity peaks within 24 h after inoculation (HAI). Ascorbate peroxidase activity was higher only in GLS-infected leaves at 6 HAI, while superoxide dismutase remained unaltered. A lower level of hydrogen peroxide (H2O2) was determined in GLS-infected plants at 48 HAI, but the electrolyte leakage markedly increased. Disease symptoms in leaves were only caused by GLS-isolate. Results suggest that the virulent isolate coordinately downregulates the oxidative plant defense responses enabling its successful establishment in apple leaves.

New insights into the characterization of Colletotrichum species associated with apple diseases in southern Brazil and Uruguay.

Colletotrichum species are associated with Apple bitter rot (ABR) and Glomerella leaf spot (GLS). Whereas both apple diseases occur frequently in Brazil, only the former has been reported in Uruguay. This work was aimed at identifying and comparing morpho-cultural characteristics and pathogenic variability of thirty-nine Colletotrichum isolates from both countries. Sequencing of the internal transcribed spacer (ITS) rDNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and ß-tubulin (TUB2) allowed the identification of three species causing ABR and GLS in Brazil, i.e., Colletotrichum fructicola, Colletotrichum karstii, and Colletotrichum nymphaeae; and three species causing ABR in Uruguay, i.e., C. fructicola, Colletotrichum theobromicola, and Colletotrichum melonis. Six groups of colony colours were recorded with group 1 (mycelium white to pink and in reverse pinkish) and group 2 (mycelium white to grey and in reverse pinkish) the most frequent. Isolates of C. fructicola and C. theobromicola were sensitive to benomyl, while C. karstii, C. nymphaeae, and C. melonis were resistant. Conidia were predominantly cylindrical for C. fructicola and C. karstii, fusiform for C. nymphaeae and C. melonis, and obclavate for C. theobromicola. Brazilian isolates caused ABR in wounded fruits, but only five in non-wounded ones. Uruguayan isolates produced symptoms in fruits with or without previous wounding. All Brazilian isolates from GLS and twelve from ABR were able to cause GLS symptoms, while a sole Uruguayan ABR-isolate caused leaf spot symptoms. This study gives a better insight on the new species causing apple disease in both countries and discusses their pathogenic potential.