GAR-transferase contributes to purine synthesis and mitochondrion function to maintain fungal development and full virulence of Penicillium digitatum.

Penicillium digitatum is one of the most critical phytopathogens during the citrus postharvest period. However, the molecular mechanism of pathogenesis remains to be further explored. Purine is a multiple functional substance in organisms. To verify the role of the de novo purine biosynthesis (DNPB) pathway in P. digitatum, we investigated the third gene Pdgart, glycinamide ribonucleotide (GAR)-transferase, of this pathway in this study. The deletion mutant ΔPdgart was generated in the principle of homologous recombination via Agrobacterium tumefaciens-mediated transformation (ATMT). The phenotypic assay indicated that the ΔPdgart mutant displayed severe defects in hyphae growth, conidiation and germination, which can be rescued by the addition of exogenous ATP and AMP. Compared with wild-type strain N1, the ATP level of strain ΔPdgart was detected to be sharply declined during conidial germination, and this was resulted from the damage to purine synthesis and aerobic respiration. The pathogenicity assay suggested that mutant ΔPdgart infected citrus fruit but attenuated disease, which was owing to its reduced production of organic acids and activities of cell wall degradation enzymes. Additionally, the ΔPdgart mutant showed altered sensitivity to stress agents and fungicides. Taken together, the present study provides insights into the essential functions of Pdgart, and paves the way for further study and novel fungicide development.

Biocontrol Ability and Action Mechanism of Meyerozyma guilliermondii 37 on Soft Rot Control of Postharvest Kiwifruit.

Postharvest soft rot of kiwifruit has resulted in substantial market losses, yet there were few antagonistic yeasts reported to control the disease. This study screened 1113 yeast strains for potential antagonistic yeast to control soft rot of kiwifruit caused by Botryosphaeria dothidea and Diaporthe actinidiae, and strain 37 was selected to evaluate the control efficacy and mechanisms, which was identified as Meyerozyma guilliermondii via molecular biological identification. Our results showed that M. guilliermondii 37 effectively reduced pathogen spore germination rate to 28.52% and decay incidence of inoculated kiwifruit to 42.11% maximumly, whereas cell-free supernatant lacked antifungal activity, implying that M. guilliermondii 37 didn't produce direct antifungal compounds against the two pathogens. In addition, M. guilliermondii 37 adhered tenaciously to the pathogens' mycelium and colonized rapidly in kiwifruit flesh. Moreover, yeast strain 37 induced kiwifruit resistance by elevating the defense-related enzyme activity, increasing the antioxidant substances content, and suppressing the cell wall-degrading enzyme activity. Gene expression was consistent with the corresponding enzyme activity. Further postharvest yeast immersion treatment significantly reduced natural decay to 35.69% while maintaining soft-ripe quality. These results indicated that M. guilliermondii 37 might serve as a biocontrol agent against postharvest soft rot in kiwifruit.

Functional identification of the DHN melanin synthesis gene cluster and its role in UV-C tolerance in citrus postharvest pathogenic fungus Penicillium digitatum.

The filamentous fungus Penicillium digitatum brings out great losses in citrus fruits by causing citrus green mold disease during the postharvest period. Previously, we obtained a T-DNA insertion mutant N2130 of P. digitatum, which produced albino conidia. To understand the role of green-grey conidial pigment in P. digitatum, we identified the insertion site and deeply explored the 1,8-dihydroxynaphtsalene (DHN)-melanin synthesis gene cluster in this phytopathogen. In this study, we deleted five genes in P. digitatum, PdPksP, PdAbr1, PdArp1, PdArp2, and PdAyg1, and the experiments were further performed on phenotype analyses, including pigmentation, UV-C tolerance, virulence, growth rate, conidiation, stress (osmotic-, oxidative-, cell wall disturbing-, and high temperature-) tolerance, fungicide resistance, and conidial hydrophobicity. The results showed that the five deletion mutants (ΔPdPksP, ΔPdAbr1, ΔPdArp1, ΔPdArp2 and ΔPdAyg1) produced albino, brownish, brown, reddish-brown, and Yellowish green conidia, respectively. In addition, the survival colony forming units (CFUs) of the deletion mutants, under the treatment of UV-C radiation (261.4 mJ/cm2), were 0.3- to 0.6-fold of those surviving in wild-type strain N1. Moreover, after 522.8 mJ/cm2-UV-C-irradiation on conidia, the deletion mutants showed a larger decrease in pathogenicity on Valencia Orange fruits compared with strain N1. However, there were no significant differences among other phenotypes tested in this study. Collectively, our research reported the DHN-melanin synthesis pathway in P. digitatum for the first time, and revealed that DHN-melanin is important for P. digitatum to tolerate UV-C irradiation.

Natamycin as a safe food additive to control postharvest green mould and sour rot in citrus.

AIMS: The purpose of this study was to explore the potential inhibitory mechanism and assess the feasibility of natamycin as an antifungal agent in the utilization of citrus storage. METHODS AND RESULTS: In this study, the mycelial growth, spore germination as well as germ tube elongations of Geotrichum citri-aurantii and Penicillium digitatum were significantly inhibited by natamycin treatment. The relative conductivities of G. citri-aurantii and P. digitatum mycelia were increased as time went by and the damages of plasma membranes were up to 17.43% and 28.61%. The mitochondria abnormalities and vacuolation were also observed in the TEM. Moreover, the sour rot and green mould decay incidences were reduced to 18.33% and 10% post incubation with G. citri-aurantii and P. digitatum under 300 mg L-1 natamycin application, respectively. For the citrus storage experiment, there was no significant difference in edible rate, juice yield, total soluble solid (TSS) content, titratable acid (TA) and decay incidences of the 'Newhall' navel orange fruit treated with 300 mg L-1 natamycin stored for 90 d. CONCLUSIONS: Natamycin could decrease the expansions of green mould and sour rot and maintain quality and improve storability on citrus fruit. SIGNIFICANCE AND IMPACT OF THE STUDY: This work explores the potential inhibition mechanism of natamycin G. citri-aurantii and P. digitatum and assesses the feasibility of natamycin as an antifungal agent in the utilization of citrus storage.

Chromosome Genome Sequencing and Comparative Transcriptome-Based Analyses of Kloeckera apiculata 34-9 Unveil the Potential Biocontrol Mechanisms Against Citrus Green Mold.

Biological control is an environmentally friendly, safe, and replaceable strategy for disease management. Genome sequences of a certain biocontrol agent could lay a solid foundation for the research of molecular biology, and the more refined the reference genome, the more information it provides. In the present study, a higher resolution genome of Kloeckera apiculata 34-9 was assembled using high-throughput chromosome conformation capture (Hi-C) technology. A total of 8.07 M sequences of K. apiculata 34-9 genome was anchored onto 7 pesudochromosomes, which accounting for about 99.51% of the whole assembled sequences, and 4,014 protein-coding genes were annotated. Meanwhile, the detailed gene expression changes of K. apiculata 34-9 were obtained under low temperature and co-incubation with Penicillium digitatum treatments, respectively. Totally 254 differentially expressed genes (DEGs) were detected with low temperature treatment, of which 184 and 70 genes were upregulated and downregulated, respectively. Some candidate genes were significantly enriched in ribosome biosynthesis in eukaryotes and ABC transporters. The expression of gene Kap003732 and Kap001595 remained upregulated and downregulated through the entire time-points, respectively, indicating that they might be core genes for positive and negative response to low temperature stress. When co-incubation with P. digitatum, a total of 2,364 DEGs were found, and there were 1,247 upregulated and 1,117 downregulated genes, respectively. Biosynthesis of lysine and arginine, and phenylalanine metabolism were the highest enrichment of the cluster and KEGG analyses of the co-DEGs, the results showed that they might be involved in the positive regulation of K. apiculata 34-9 response to P. digitatum. The completeness of K. apiculata 34-9 genome and the transcriptome data presented here are essential for providing a high-quality genomic resource and it might serve as valuable molecular properties for further studies on yeast genome, expression pattern of biocontrol system, and postharvest citrus storage and preservation.

Genome sequencing and transcriptome analysis of Geotrichum citri-aurantii on citrus reveal the potential pathogenic- and guazatine-resistance related genes.

Sour rot, caused by Geotrichum citri-aurantii, is a major postharvest disease of citrus，and it causes serious economic losses. In this study, a high-quality genome sequence of G. citri-aurantii was obtained by Single Molecule Real-Time Sequencing (SMRT). Approximately 5.43 Gb of clean data were obtained and a total of 27.94-Mb genomic sequence was mapped to 10 chromosome groups after high-through chromosome conformation capture (Hi-C) assembly. In addition, three polygalacturonase genes which were related to pathogenicity in G. citri-aurantii genome were discovered. And transcriptome data of guazatine-resistance had been analyzed, the results showed that the guazatine-resistance of G. citri-aurantii was related to two ATP-binding cassette (ABC) transporter family genes, six major facilitator superfamily (MFS) transporter family genes and two multidrug and toxic compound extrusion (MATE) transporter family genes. In summary, our research may provide novel insights into the effective control of this pathogen.

Genome-wide identification, characterization and expression analysis of lineage-specific genes within Hanseniaspora yeasts.

Lineage-specific genes (LSGs) are defined as genes with sequences that are not significantly similar to those in any other lineage. LSGs have been proposed, and sometimes shown, to have significant effects in the evolution of biological function. In this study, two sets of Hanseniaspora spp. LSGs were identified by comparing the sequences of the Kloeckera apiculata genome and of 80 other yeast genomes. This study identified 344 Hanseniaspora-specific genes (HSGs) and 109 genes ('orphan genes') specific to K. apiculata. Three thousand three hundred thirty-one K. apiculata genes that showed significant similarity to at least one sequence outside the Hanseniaspora were classified into evolutionarily conserved genes. We analyzed their sequence features, functional categories, gene origin, gene structure and gene expression. We also investigated the predicted cellular roles and Gene Ontology categories of the LSGs using functional inference. The patterns of the functions of LSGs do not deviate significantly from genome-wide average. The results showed that a few LSGs were formed by gene duplication, followed by rapid sequence divergence. Many of the HSGs and orphan genes exhibited altered expression in response to abiotic stress. Studying these LSGs might be helpful for understanding the molecular mechanism of yeast adaption.

The Shared and Specific Genes and a Comparative Genomics Analysis within Three Hanseniaspora Strains.

Kloeckera apiculata plays an important role in the inhibition of citrus postharvest blue and green mould diseases. This study was based on the previous genome sequencing of K. apiculata strain 34-9. After homologous comparison, scaffold 27 was defined as the mitochondrial (mt) sequence of K. apiculata 34-9. The comparison showed a high level of sequence identity between scaffold 27 and the known mtDNA of Hanseniaspora uvarum. The genome sequence of H. vineae T02/19AF showed several short and discontinuous fragments homologous to the mtDNA of H. uvarum. The shared and specific genes of K. apiculata, H. uvarum, and H. vineae were analysed by family using the TreeFam methodology. GO analysis was used to classify the shared and specific genes. Most of the gene families were classified into the functional categories of cellular component and metabolic processes. The whole-genome phylogram and genome synteny analysis showed that K. apiculata was more closely related to H. uvarum than to H. vineae. The genomic comparisons clearly displayed the locations of the homologous regions in each genome. This analysis could contribute to discovering the genomic similarities and differences within the genus Hanseniaspora. In addition, some regions were not collinearity-matched in the genome of K. apiculata compared with that of H. uvarum or H. vineae, and these sequences might have resulted from evolutionary variations.

Development of Penicillium italicum-Specific Primers for Rapid Detection among Fungal Isolates in Citrus.

Blue mold in citrus is caused by Penicillium italicum. In this study, the P. italicum-specific primers were developed for rapid detection based on the conserved genes RPB1 and RPB2 among Penicillium genomes. The two primer pairs RPB1-a and RPB1-b proved to be specific to detect P. italicum. The PCR assay among 39 fungal isolates and the colonial, pathogenic morphologies and molecular methods validated the specificity and reliability of these two primer pairs. This report provided a method and P. italicum-specific primers, which might greatly contribute to citrus postharvest industry.

Antagonistic Activity and the Mechanism of Bacillus amyloliquefaciens DH-4 Against Citrus Green Mold.

Citrus fruit usually suffer significant losses during the storage and transportation stages. Green mold, a postharvest rot of citrus fruit caused by Penicillium digitatum, is one of the most serious fungal diseases. In this study, the antagonist strain DH-4 was identified as Bacillus amyloliquefaciens according to morphological observation and 16S ribosomal DNA analysis. In addition, it showed broad antifungal activity, especially the suppression of Penicillium spp. The culture filtrate of strain DH-4 exhibited apparent activity against P. digitatum in vitro and in vivo. In storage, the culture filtrate with DH-4 in it showed a better antiseptic effect. The antifungal substances in the culture filtrate, produced by strain DH-4, displayed stable activity in various extreme conditions. In addition, the antifungal substances in the culture filtrate were identified as macrolactin, bacillaene, iturins, fengycin, and surfactin by ultraperformance liquid chromatography (UPLC) electrospray ionization mass spectrometry analysis. The UPLC fractions containing these antifungal compounds were basically heat tolerant and all responsible for the antagonistic activity against P. digitatum. Transmission electron microscope observation indicated that the antifungal substances might cause abnormalities in the P. digitatum cellular ultrastructure, which could be the possible mode of action of B. amyloliquefaciens against P. digitatum. In addition, it was confirmed via scanning electron microscope analysis that the main way it inhibited P. digitatum was by secreting antimicrobial compounds without direct interaction. This study contributes to the understanding of the mechanism of B. amyloliquefaciens against citrus green mold as well as providing a potential application for the biocontrol of postharvest rot diseases in citrus fruit.

Comparative transcriptional profiling of orange fruit in response to the biocontrol yeast Kloeckera apiculata and its active compounds.

BACKGROUND: The yeast Kloeckera apiculata strain 34-9 is an antagonist that shows biological control activity against the postharvest fungal pathogens of citrus. An antifungal compound, 2-phenylethanol (PEA), has been identified from the extract of K. apiculata. To better understand the molecular processes underlying the response of citrus fruit tissue to K. apiculata, the extract and PEA, microarray analyses were performed on navel oranges using an Affymetrix Citrus GeneChip. RESULTS: As many as 801, 339 and 608 differentially expressed genes (DEGs) were identified after the application of K. apiculata, the extract and PEA, respectively. In general, K. apiculata induced the expression of defence-related genes. In addition to chitinase and ß-1,3-glucanase, genes involved in ethylene (ET), jasmonic acid (JA), calcium signalling, MAPK signalling and phenylalanine metabolism were induced. In contrast, monodehydroascorbate reductase, superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) and carotenoid biosynthesis genes were down-regulated. The expression profiles for the extract- and PEA-treated samples were similar to that found for yeast (sharing 57.4 % DEGs), with a significant increase in the transcript levels of defence-related genes. CONCLUSION: This study provides a global picture of the gene expression changes in navel oranges after the application of the antagonist yeast K. apiculata, its extract and PEA. The interpretation of the DEGs revealed new insight into the molecular processes that regulate the defence responses in orange tissue.

Characterization of the expression of the stress-responsive PpERS1 gene from peach and analysis of its promoter using transgenic tomato.

The PpERS1 gene, which encodes an ethylene receptor and responds to abiotic and biotic stresses, was cloned from peach (Prunus persica L. Batsch cv Okubao). The genomic DNA sequence of PpERS1 comprises seven exons which are separated by six introns, interestingly alternative splicing of the first intron produced three different PpERS1 transcripts. In addition, a 2.8-kb sequence including the promoter of PpERS1 was isolated and analyzed by placing expressing of the GUS reporter gene under its control. Several putative cis-elements were identified in the promoter of PpERS1, including two ethylene-responsive elements (EREs), five W boxes, and four putative binding sites for MYB-type transcription factors. Deletion analysis indicated the presence of an enhancer element in the PpERS1 promoter. Temporal and spatial expression analysis of the PpERS1 promoter using histochemical GUS staining showed GUS activity in all tissues examined throughout the development of transgenic tomato plants. Exposure to various stresses caused similar changes in expression patterns in peach and transgenic tomato plants. Overall, our results suggested that PpERS1 gene might play important roles in response to multiple stresses via signal transduction mediated by ethylene receptors. The characterization of the PpERS1 promoter contributes to our understanding of the transcriptional regulation of this ethylene receptor in peach.

Mechanisms of action for 2-phenylethanol isolated from Kloeckera apiculata in control of Penicillium molds of citrus fruits.

BACKGROUND: Green and blue mold decay, caused by Penicillium digitatum and P. italicum, respectively, are important postharvest diseases of citrus. Biocontrol by microbes is an alternative to synthetic fungicide application. In this study, the antagonistic yeast strain Kloeckera apiculata 34-9 was used to investigate the action mechanisms involved in the biocontrol of postharvest diseases. RESULTS: An antifungal substance, 2-phenylethanol (PEA), was isolated from K. apiculata and demonstrated to have antimicrobial activity against selected phytopathogenic fungi. Experiments on P. italicum cells identified the mitochondria and the nucleus as particularly sensitive to inhibition. Regulation of P. italicum gene expression was investigated using RNA-Seq. PEA up-regulated genes involved with the peroxisome, regulation of autophagy, phosphatidylinositol signaling system, protein processing in endoplasmic reticulum, fatty acid metabolism, and inhibited ribosome, RNA polymerase, DNA replication, amino acid biosynthesis, aminoacyl-tRNA biosynthesis and cell cycle. Inhibitory responses revealed by RNA-Seq suggest that PEA might compete for attachment on the active site of phenylalanyl-tRNA synthetase (PheRS). CONCLUSION: This study provided new insight on the mode of action of biocontrol yeast agents in controlling postharvest pathogenic fungi.

Farnesol induces apoptosis and oxidative stress in the fungal pathogen Penicillium expansum.

This study was conducted to evaluate the effect of farnesol (FOH) on the growth of P. expansum. The viability of P. expansum cells was determined by counting the colony forming units (CFU) after each FOH treatment. Morphological changes of FOH-treated fungal cells were analyzed by staining with Hoechst 33258, TUNEL (terminal deoxynucleotidyl transferase fluorescein-12-dUTP nick end labeling), Annexin-V FITC and the oxidant-sensitive probe H2DCFDA (dichlorodihydro-fluorescein diacetate). FOH strongly inhibited the growth of hyphae. The hyphal cells showed the hallmarks of apoptosis including chromatin condensation, DNA fragmentation, phosphatidylserine (PS) externalization, caspases activation, intracellular reactive oxygen species (ROS) generation but without nucleosomal ladder production. The abnormal cellular ultrastructure observed by transmission electron microscope (TEM) indicated that disintegration of cellular ultrastructure (especially for mitochondria) was linked to FOH-induced cell death. Taken together we demonstrated that FOH inhibits the growth of P. expansum and promotes apoptosis via activation of metacaspases, production of ROS and disintegration of cellular ultrastructure.