Isolation and Characterization of Bacillus velezensis Strain P2-1 for Biocontrol of Apple Postharvest Decay Caused by Botryosphaeria dothidea

Botryosphaeria dothidea causes apple ring rot, which is among the most prevalent postharvest diseases of apples and causes significant economic loss during storage. In this study, we investigated the biocontrol activity and possible mechanism of Bacillus velezensis strain P2-1 isolated from apple branches against B. dothidea in postharvest apple fruit. The results showed strain P2-1, one of the 80 different endophytic bacterial strains from apple branches, exhibited strong inhibitory effects against B. dothidea growth and resulted in hyphal deformity. B. velezensis P2-1 treatment significantly reduced the ring rot caused by B. dothidea. Additionally, the supernatant of strain P2-1 exhibited antifungal activity against B. dothidea. Re-isolation assay indicated the capability of strain P2-1 to colonize and survive in apple fruit. PCR and qRT-PCR assays revealed that strain P2-1 harbored the gene clusters required for biosynthesis of antifungal lipopeptides and polyketides. Strain P2-1 treatment significantly enhanced the expression levels of pathogenesis-related genes (MdPR1 and MdPR5) but did not significantly affect apple fruit qualities (measured in fruit firmness, titratable acid, ascorbic acid, and soluble sugar). Thus, our results suggest that B. velezensis strain P2-1 is a biocontrol agent against B. dothidea-induced apple postharvest decay. It acts partially by inhibiting mycelial growth of B. dothidea, secreting antifungal substances, and inducing apple defense responses.

Transcriptome Analysis Reveals the Inducing Effect of Bacillus siamensis on Disease Resistance in Postharvest Mango Fruit

Postharvest anthracnose, caused by the fungus Colletotrichum gloeosporioides, is one of the most important postharvest diseases of mangoes worldwide. Bacillus siamensis (B. siamensis), as a biocontrol bacteria, has significant effects on inhibiting disease and improving the quality of fruits and vegetables. In this study, pre-storage application of B. siamensis significantly induced disease resistance and decreased disease index (DI) of stored mango fruit. To investigate the induction mechanisms of B. siamensis, comparative transcriptome analysis of mango fruit samples during the storage were established. In total, 234,808 unique transcripts were assembled and 56,704 differentially expressed genes (DEGs) were identified by comparative transcriptome analysis. Gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of DEGs showed that most of the DEGs involved in plant-pathogen interaction, plant hormone signal transduction, and biosynthesis of resistant substances were enriched. Fourteen DEGs related to disease-resistance were validated by qRT-PCR, which well corresponded to the FPKM value obtained from the transcriptome data. These results indicate that B. siamensis treatment may act to induce disease resistance of mango fruit by affecting multiple pathways. These findings not only reveal the transcriptional regulatory mechanisms that govern postharvest disease, but also develop a biological strategy to maintain quality of post-harvest mango fruit.

Inhibitory Effect and Mechanism of Chitosan–Ag Complex Hydrogel on Fungal Disease in Grape

Hydrogel antibacterial agent is an ideal antibacterial material because of it could diffuses antibacterial molecules into the decayed area by providing a suitable microenvironment and the hydrogel acts as a protective barrier on the decay interface. The biocompatibility and biodegradation make the removal process easily which were widely used in medical fields. However, there have been few reports on its application for controlling postharvest diseases in fruit. In this study, the Chitosan-Ag (CS-Ag) complex hydrogels were prepared using the physical crosslinking method, which used for controlling postharvest diseases in grape. The prepared hydrogels were stable for a long period at room temperature. The structure and surface morphology of CS-Ag composite hydrogels were characterized by UV-Vis, FTIR, SEM, and XRD. The inhibitory effects of CS-Ag hydrogel on disease in grape caused by P. expansum, A. niger and B. cinerea were investigated both in vivo and in vitro. The remarkable antibacterial activity of CS-Ag hydrogels was mainly due to the synergistic antibacterial and antioxidant effects of CS and Ag. Preservation test showed that the CS-Ag hydrogel had positive fresh-keeping effect. This revealed CS-Ag hydrogels plays a critical role in controlling fungal disease in grape.

Fungicide Resistance in Alternaria alternata from Blueberry in California and Its Impact on Control of Alternaria Rot

Alternaria rot caused by Alternaria alternata is one of the major postharvest diseases affecting blueberries in California. The sensitivity profiles of A. alternata from blueberry field to quinone outside inhibitors (QoIs), boscalid, fluopyram, fludioxonil, cyprodinil and polyoxin D in California were examined in this study. EC50 values of 51 A. alternata isolates for boscalid varied greatly among the isolates, ranging from 0.265 to >100 µg/ml. EC50 values of 51 A. alternata isolates to fluopyram, fludioxonil, cyprodinil, and polyoxin D were 5.188 ± 7.118 µg/ml, 0.078 ± 0.021 µg/ml, 0.465 ± 0.302 µg/ml, and 6.238 ± 7.352 µg/ml, respectively. In total, 143 isolates were screened for resistance at 5 and 10 µg/ml for fludioxonil, cyprodinil, and fluopyram, 10 µg/ml for polyoxin D, and 10 and 50 µg/ml for boscalid. Based on the published discriminatory concentrations for phenotyping resistance, of the 143 isolates, all were considered resistant to boscalid; 32, 69 and 42 were sensitive, low resistant, and resistant to fluopyram, respectively; and all were sensitive to fludioxonil and cyprodinil. In a PCR-RFLP method for phenotyping, 60 out of the 143 isolates were classified as resistant to QoIs. Control tests on detached blueberry fruit inoculated with different Alternaria isolates showed that fludioxonil and cyprodinil significantly reduced disease incidence and severity; however, pyraclostrobin, boscalid, fluopyram and polyoxin D significantly reduced only disease severity. The obtained results will be helpful in making decisions on fungicide programs to control A. alternata isolates with resistance or reduced sensitivities to multiple fungicides.

A Sustainable Alternative for Postharvest Disease Management and Phytopathogens Biocontrol in Fruit: Antagonistic Yeasts

Postharvest diseases of fruits caused by phytopathogens cause losses up to 50% of global production. Phytopathogens control is performed with synthetic fungicides, but the application causes environmental contamination problems and human and animal health in addition to generating resistance. Yeasts are antagonist microorganisms that have been used in the last years as biocontrol agents and in sustainable postharvest disease management in fruits. Yeast application for biocontrol of phytopathogens has been an effective action worldwide. This review explores the sustainable use of yeasts in each continent, the main antagonistic mechanisms towards phytopathogens, their relationship with OMIC sciences, and patents at the world level that involve yeast-based-products for their biocontrol.

Incidence, Speciation, and Morpho-Genetic Diversity of Penicillium spp. Causing Blue Mold of Stored Pome Fruits in Serbia

Blue mold, caused by Penicillium spp., is one of the most economically important postharvest diseases of pome fruits, globally. Pome fruits, in particular apple, is the most widely grown pome fruit in Serbia, and the distribution of Penicillium spp. responsible for postharvest decay is unknown. A two-year survey was conducted in 2014 and 2015, where four pome fruits (apple, pear, quince, and medlar) with blue mold symptoms were collected from 20 storage locations throughout Serbia. Detailed morphological characterization, analysis of virulence in three apple cultivars, and multilocus phylogeny revealed three main Penicillium spp. in order of abundance: P. expansum, P. crustosum, and P. solitum. Interestingly, P. expansum split into two distinct clades with strong statistical support that coincided with several morphological observations. Findings from this study are significant and showed previously undocumented diversity in blue mold fungi responsible for postharvest decay including the first finding of P. crustosum, and P. solitum as postharvest pathogens of quince and P. crustosum of medlar fruit in the world, and P. expansum of quince in Serbia. Data from this study provide timely information regarding phenotypic, morphological and genotypic plasticity in P. expansum that will impact the design of species-specific detection tools and guide the development of blue mold management strategies.

Identification of Volatile Organic Compounds in Extremophilic Bacteria and Their Effective Use in Biocontrol of Postharvest Fungal Phytopathogens

Phytopathogenic fungal growth in postharvest fruits and vegetables is responsible for 20–25% of production losses. Volatile organic compounds (VOCs) have been gaining importance in the food industry as a safe and ecofriendly alternative to pesticides for combating these phytopathogenic fungi. In this study, we analysed the ability of some VOCs produced by strains of the genera Bacillus, Peribacillus, Pseudomonas, Psychrobacillus and Staphylococcus to inhibit the growth of Alternaria alternata, Botrytis cinerea, Fusarium oxysporum, Fusarium solani, Monilinia fructicola, Monilinia laxa and Sclerotinia sclerotiorum, in vitro and in vivo. We analysed bacterial VOCs by using GC/MS and 87 volatile compounds were identified, in particular acetoin, acetic acid, 2,3-butanediol, isopentanol, dimethyl disulphide and isopentyl isobutanoate. In vitro growth inhibition assays and in vivo experiments using cherry fruits showed that the best producers of VOCs, Bacillus atrophaeus L193, Bacillus velezensis XT1 and Psychrobacillus vulpis Z8, exhibited the highest antifungal activity against B. cinerea, M. fructicola and M. laxa, which highlights the potential of these strains to control postharvest diseases. Transmission electron microscopy micrographs of bacterial VOC-treated fungi clearly showed antifungal activity which led to an intense degeneration of cellular components of mycelium and cell death.

Application of Rosemary and Eucalyptus Essential Oils and Their Main Component on the Preservation of Apple and Pear Fruits

Nowadays, increase fruit losses are being reported due to the development of fungal postharvest diseases. In an attempt to reduce the use of synthetic fungicides, a turn towards natural products such as essential oils (EOs) and natural compounds has been made. The objective of this study was to investigate the effects of eucalyptus (Euc), rosemary (Ros) EO, their mixture (50:50 v/v) and their common main component (i.e., eucalyptol) on the quality parameters, fruit response and inhibition of blue rot (Penicillium expansum) in apple and pear fruits during their shelf life. The results of the present study revealed that fungal colony growth decreased in vitro with exposure at eucalyptus EO (Euc-300 μL/L), rosemary EO (Ros-300 μL/L) and their mixture (Euc + Ros 100 and 300 μL/L). The exposure at Ros-100 μL/L stimulated spore production, whilst Euc + Ros (100 and 300 μL/L) and eucalyptol (100 and 300 μL/L) decreased spore germination. Moreover, the in vivo applied treatments resulted in decreased lesion growth of P. expansum in apple and pear fruits. Respiration rate increased with the application of Euc + Ros at 300 μL/L and eucalyptus EO (Euc-100 μL/L and Euc-300 μL/L) for both assessed fruits. On the other hand, no significant differences were reported on apples and pears total soluble solids and acidity values. The application of Euc + Ros-300 μL/L in apples increased hydrogen peroxide (H2O2) levels, whilst Euc-100 and Euc-300 μL/L increased lipid peroxidation levels. Regarding pear fruits, exposure to Euc-100 μL/L and Ros-100 μL/L resulted in increased H2O2 whereas, Euc-100 μL/L, Ros- (100 and 300 μL/L) and eucalyptol (100 and 300 μL/L) also increased lipid peroxidation. The findings of this study indicate that the investigated natural products can be explored for the preservation of fresh apples and pears, as alternative natural fungicides with consideration of the fresh produce quality attributes.

Stability of Dry and Liquid Metschnikowia pulcherrima Formulations for Biocontrol Applications against Apple Postharvest Diseases

The yeast Metschnikowia pulcherrima is frequently isolated from environmental samples and has often been reported to exhibit strong antagonistic activity against plant pathogens. In order to assess the potential of this species for its development into a plant protection product, the survival during formulation and storage were quantified and field efficacy was assessed over a period of five years. Freeze dried and liquid M. pulcherrima formulations (i.e., with skim milk powder (SMP), sucrose, glycerol, xanthan, without additives) were prepared and the number of viable cells was quantified during storage at different temperatures. Field trials against apple postharvest diseases (Neofabreae) were performed with different dry formulations. M. pulcherrima proved exceptionally stable for many months and even years. Five years of field trials with the yeast revealed variable effects, but reduced Neofabreae infections of stored apples were observed in some years. M. pulcherrima applications after prior fungicide treatments repeatedly showed an additive effect as compared to the fungicide treatments alone. In summary, M. pulcherrima exhibited highly advantageous storage properties and encouraging activity against apple postharvest rots. Further studies to identify the factors responsible for antagonistic activity in the field and survival during storage are expected to lay the foundation for the future development of a plant protection product.