First Report of Fruit Rot of Sweet Pepper Caused by Cladosporium cladosporioides in Israel.

Sweet pepper (Capsicum annuum L.) is an important vegetable crop grown in Israel for local and export markets. Bell pepper 'Dinamo' and sweet bite pepper exhibiting fruit spotting were observed in postharvest storage of commercial farms in Giv'at Ko'ah (32°02'21″ N; 34°56'41″ E) and Arava (30°46'41″ N; 35°14'28″ E) in July, 2021. Fruit with lesions were sporadic at each facility, however, limited numbers of packaged boxes were discarded due to a high percentage of symptomatic fruit. The spots appeared as slightly sunken dark brown or black lesions, coated by a fine velvety layer of olive-grey conidia (Suppl. Fig. 1). A fungus was routinely isolated from the fruit onto potato dextrose agar amended with 50 ppm chloramphenicol (PDAC) and was tentatively identified as a Cladosporium spp. based on colony morphology and conidial appearance (Suppl. Fig. 1). Isolates (n = 6) of the fungus were cultured on malt extract agar (MEA) and PDA for morphological observation. On MEA, the pathogen sporulated profusely and conidia measured 4.5 (3 - 7) × 3.5 (2 - 4) µm and were smooth and globose or sub-globose. Ramoconidia had 0 or 1 septations and measured 13.4 (10 - 18) × 3.9 (3 - 6) µm. Conidiophores were solitary, straight or flexous, usually unbranched, and measured 60 - 350 µm. Both macro- and micronematous conidiophores were present. Hyphae were flat and immersed, olivaceous-grey, septate and rope-like. The fungus was slow growing, reaching 41.0 mm after 10 d incubation at 20°C. To confirm species identity, the isolates were grown in potato dextrose broth and DNA was extracted from the mycelium using MasterPure DNA Purification Kit (EpiCentre, Madison, WI, USA) following the manufacturer's instructions. The internal transcribed spacer (ITS) region was amplified using primers ITS 1 and ITS 4, and elongation factor-1 and actin gene regions with EF1-728F/986R and ACT-512F/783R, respectively (Carbone and Kohn, 1999). The PCR products were sequenced and a BLASTn search showed 99-100% similarity to Cladosporium cladosporioides (GenBank accession nos. MW255614.1, MK416093.1, MK306457.1), a common fungal species found worldwide as a plant pathogen and saphrophyte (Bensch et al., 2012). The ITS gene sequences for a representative isolate from each location were deposited in GenBank (accession nos. OK104139.1 and OL672241.1). To confirm pathogenicity, red bell pepper fruits (n = 8) were wounded with a sterile needle and a 12 µl drop of conidial suspension (4 x 105/ml) of each isolate was pipetted onto the wound. Control fruit received sterile water. The fruit were incubated at 100% RH in sealed containers at 20-22°C. After 7-10 d incubation small dark sunken spots developed, and by 14 d lesions formed with velvety conidial growth similar to the ones originally observed. The fungus was successfully isolated again from the lesions onto PDA and confirmed as C. cladosporioides based on colony and conidial morphology. No symptoms developed on fruit inoculated with sterile water. The pathogenicity test was repeated two times with similar results. C. cladosporioides is a cosmopolitan pathogen and has been cited causing disease on numerous crops worldwide (Bensch et al., 2012). This species was reported causing tomato leaf spot in Mexico (Robles-Yerena et al., 2019) and black mould of tomato fruit in Australia (Ma et al., 2020). Cladosporium herbarum was found causing fruit rot of bell pepper in the U.S. (Ramsey and Heiberg, 1952). Sweet peppers are widely grown in Israel, and have high economic value as an export crop. Control measures to limit losses from C. cladosporioides postharvest may be necessary if conditions are favorable for disease during prolonged storage and refrigerated transport.

Identification and Functional Analysis of NLP-Encoding Genes from the Postharvest Pathogen Penicillium expansum.

Penicillium expansum is a major postharvest pathogen that infects different fruits, mainly through injuries inflicted during harvest or subsequent handling after harvest. Several effectors were suggested to mediate pathogenicity of P. expansum in fruit tissue. Among these effectors Nep1-like proteins (NLPs), produced by various microorganisms with different lifestyles, are known for their ability to induce necrosis in dicot plants and were shown to be involved in virulence of several plant-related pathogens. This study was aimed at the identification and functional characterization of two NLP genes found in the genome of P. expansum. The genes were designated Penlp1 and Penlp2 and were found to code type1 and type3 NLP respectively. Necrosis-inducing activity of the two proteins was demonstrated by transient expression in Nicotiana benthamiana leaves. While Penlp1 expression was induced during apple infection and in liquid culture, the highest level of Penlp2 expression was found in ungerminated spores. Deletion of Penlp1, but not Penlp2, resulted in reduced virulence on apples manifested by reduced rate of lesion development (disease severity).

Identification and characterization of LysM effectors in Penicillium expansum.

P. expansum is regarded as one of the most important postharvest rots of apple fruit and is also of great concern to fruit processing industries. Elucidating the pathogenicity mechanism of this pathogen is of utmost importance for the development of effective and safe management strategies. Although, many studies on modification of the host environment by the pathogen were done, its interactions with fruit during the early stages of infection and the virulence factors that mediate pathogenicity have not been fully defined. Effectors carrying LysM domain have been identified in numerous pathogenic fungi and their role in the first stages of infection has been established. In this study, we identified 18 LysM genes in the P. expansum genome. Amino acid sequence analysis indicated that P. expansum LysM proteins belong to a clade of fungal-specific LysM. Eleven of the discovered LysM genes were found to have secretory pathway signal peptide, among them, 4 (PeLysM1 PeLysM2, PeLysM3 and PeLysM4) were found to be highly expressed during the infection and development of decay of apple fruit. Effect of targeted deletion of the four putative PeLysM effectors on the growth and pathogenicity was studied. Possible interactions of PeLysM with host proteins was investigated using the yeast-two-hybrid system.

Global changes in gene expression of grapefruit peel tissue in response to the yeast biocontrol agent Metschnikowia fructicola.

To gain a better understanding of the molecular changes taking place in citrus fruit tissue following the application of the yeast biocontrol agent Metschnikowia fructicola, microarray analysis was performed on grapefruit surface wounds using an Affymetrix Citrus GeneChip. Using a cut-off of P < 0.05 and a 1.5-fold change difference as biologically significant, the data indicated that 1007 putative unigenes showed significant expression changes following wounding and yeast application relative to wounded controls. Microarray results of selected genes were validated by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR). The data indicated that yeast application induced the expression of the genes encoding Respiratory burst oxidase (Rbo), mitogen-activated protein kinase (MAPK) and mitogen-activated protein kinase kinase (MAPKK), G-proteins, chitinase (CHI), phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS) and 4-coumarate-CoA ligase (4CL). In contrast, three genes, peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT), were down-regulated in grapefruit peel tissue treated with yeast cells. Moreover, suppression was correlated with significantly higher levels of hydrogen peroxide, superoxide anion and hydroxyl radical production in yeast-treated surface wounds. Interestingly, large amounts of hydrogen peroxide were detected inside yeast cells recovered from wounded fruit tissue, indicating the ability of the yeast to activate reactive oxygen species when it is in contact with plant tissue. This study provides the first global picture of gene expression changes in grapefruit in response to the yeast antagonist M. fructicola.