Survivability and behavior of probiotic bacteria encapsulated by internal gelation in non-dairy matrix and In Vitro GIT conditions.

The primary objective of this investigation was to assess the viability of free and encapsulated Lactobacillus plantarum probiotics in mango juice and under simulated gastrointestinal conditions. Specifically, the probiotics were encapsulated using sodium alginate and alginate-soy protein isolate through the internal gelation method, and the obtained probiotics were characterized for various attributes. Both free and encapsulated probiotics were exposed to challenging conditions, including thermal stress, low temperature, and simulated gastrointestinal conditions. Additionally, both types of probiotics were incorporated into mango juice, and their survival was monitored over a 28-day storage period. Following viability under simulated gastrointestinal conditions, the count of free and encapsulated probiotic cells decreased from initial levels of 9.57 log CFU/mL, 9.55 log CFU/mL, and 9.53 log CFU/mL, 9.56 log CFU/mL to final levels of 6.14 log CFU/mL, 8.31 log CFU/mL, and 6.24 log CFU/mL, 8.62 log CFU/mL, respectively. Notably, encapsulated probiotics exhibited a decrease of 1.24 log CFU and 0.94 log CFU, while free cells experienced a reduction of 3.43 log CFU and 6.24 log CFU in mango juice over the storage period. Encapsulated probiotics demonstrated higher viability in mango juice compared to free probiotics throughout the 28-day storage period. These findings suggest that mango juice can be enriched with probiotics to create a health-promoting beverage.

Biological characterization of emerging fungal pathogen Colletotrichum associated with mango (Mangifera indica L.) post-harvest anthracnose from Vietnam.

BACKGROUND: Post-harvest anthracnose (PHA) of mango is a devastating disease, which results in huge loss to mango producers and importers. Various species of PHA, diverse pathogenicity, and different resistance towards fungicides make it essential to evaluate the pathogen taxonomic status and biological characterization. METHODS AND RESULTS: Two strains DM-1 and DM-2 isolated from the fruit of DaQing mango from Vietnam were identified as Colletotrichum fructicola and C. asianum respectively, based on the morphological features, along with the phylogenetic tree of ITS and ApMat combined sequences. The growth status of different Colletotrichum strains under different conditions was analyzed to reveal the biological characteristics. The optimum growth temperature of DM-1 and DM-2 was 28 °C and mycelia grew rapidly in the dark. Both strains could grow in media with pH 4-11, while the optimum pH value was 6. Maltose and soluble starch were the most suitable carbon source for DM-1 and DM-2 respectively, and the peptone was the most suitable nitrogen source for both strains. The lethal temperatures were recorded as 55 °C 5 min for DM-1, and 50 °C 10 min for DM-2. CONCLUSIONS: To the best of our knowledge, it is the first study reporting the identification of the pathogens: C. fructicola and C. asianum responsible for postharvest fruit anthracnose of mango in Vietnam.

Simple one-step treatment for saccharification of mango peels using an optimized enzyme cocktail of Aspergillus niger ATCC 9642.

Developing efficient microbiological methods to convert polysaccharide-rich materials into fermentable sugars, particularly monosaccharides, is vital for advancing the bioeconomy and producing renewable chemicals and energy sources. This study focused on optimizing the production conditions of an enzyme cocktail from Aspergillus niger ATCC 9642 using solid-state fermentation (SSF) and assessing its effectiveness in saccharifying mango peels through a simple, rapid, and efficient one-step process. A rotatable central composite design was employed to determine optimal conditions of moisture, time, and pH for enzyme production in SSF medium. The optimized enzyme cocktail exhibited cellulase activity (CMCase) at 6.28 U/g, filter paper activity (FPase) at 3.29 U/g, and pectinase activity at 117.02 U/g. These optimal activities were achieved with an SSF duration of 81 h, pH of 4.66, and a moisture content of 59%. The optimized enzyme cocktail effectively saccharified the mango peels without the need for chemical agents. The maximum saccharification yield reached approximately 81%, indicating efficient conversion of mango peels into sugars. The enzyme cocktail displayed consistent thermal stability within the tested temperature range of 30-60°C. Notably, the highest sugar release occurred within 36 h, with glucose, arabinose, galactose, and xylose being the primary monosaccharides released during saccharification. This study highlights the potential application of Aspergillus niger ATCC 9642 and SSF for enzymatic production, offering a simple and high-performance process for monosaccharide production. The optimized enzyme cocktail obtained through solid-state fermentation demonstrated efficient saccharification of mango peels, suggesting its suitability for industrial-scale applications.

Identification of gene modules and hub genes associated with Colletotrichum siamense infection in mango using weighted gene co-expression network analysis.

Colletotrichum siamense is a hemibiotrophic ascomycetous fungus responsible for mango anthracnose. The key genes involved in C. siamense infection remained largely unknown. In this study, we conducted weighted gene co-expression network analysis (WGCNA) of RNA-seq data to mine key genes involved in Colletotrichum siamense-mango interactions. Gene modules of Turquoise and Salmon, containing 1039 and 139 respectively, were associated with C. siamense infection, which were conducted for further analysis. GO enrichment analysis revealed that protein synthesis, organonitrogen compound biosynthetic and metabolic process, and endoplasmic reticulum-related genes were associated with C. siamense infection. A total of 568 proteins had homologs in the PHI database, 370 of which were related to virulence. The hub genes in each module were identified, which were annotated as O-methyltransferase (Salmon) and Clock-controlled protein 6 (Turquoise). A total of 24 proteins exhibited characteristics of SCRPs. By using transient expression in Nicotiana benthamiana, the SCRPs of XM_036637681.1 could inhibit programmed cell death (PCD) that induced by BAX (BCL-2-associated X protein), suggesting that it may play important roles in C. siamense infection. A mango-C. siamense co-expression network was constructed, and the mango gene of XM_044632979.1 (auxin-induced protein 15A-like) was positively associated with 5 SCRPs. These findings help to deepen the current understanding of necrotrophic stage in C. siamense infection.

The evolution of mini-chromosomes in the fungal genus Colletotrichum.

Anthracnose diseases caused by Colletotrichum species are among the most common fungal diseases. These symptoms typically manifest as dark, sunken lesions on leaves, stems, and fruit. In China, mango anthracnose seriously affects fruit yield and quality. Genome sequencing of several species shows the presence of mini-chromosomes. These are thought to contribute to virulence, but their formation and activity remain to be fully elucidated. Here, we assembled 17 Colletotrichum genomes (16 isolated from mango plus one from persimmon) through PacBio long-read sequencing. Half of the assembled scaffolds had telomeric repeats at both ends indicating full-length chromosomes. Based on comparative genomics analysis at interspecies and intraspecies levels, we identified extensive chromosomal rearrangements events. We analyzed mini-chromosomes of Colletotrichum spp. and found large variation among close relatives. In C. fructicola, homology between core chromosomes and mini-chromosomes suggested that some mini-chromosomes were generated by recombination of core chromosomes. In C. musae GZ23-3, we found 26 horizontally transferred genes arranged in clusters on mini-chromosomes. In C. asianum FJ11-1, several potential pathogenesis-related genes on mini-chromosomes were upregulated, especially in strains with highly pathogenic phenotypes. Mutants of these upregulated genes showed obvious defects in virulence. Our findings provide insights into the evolution and potential relationships to virulence associated with mini-chromosomes. IMPORTANCE Colletotrichum is a cosmopolitan fungal genus that seriously affects fruit yield and quality of many plant species. Mini-chromosomes have been found to be related to virulence in Colletotrichum. Further examination of mini-chromosomes can help us elucidate some pathogenic mechanisms of Colletotrichum. In this study, we generated novel assemblies of several Colletotrichum strains. Comparative genomic analyses within and between Colletotrichum species were conducted. We then identified mini-chromosomes in our sequenced strains systematically. The characteristics and generation of mini-chromosomes were investigated. Transcriptome analysis and gene knockout revealed pathogenesis-related genes located on mini-chromosomes of C. asianum FJ11-1. This study represents the most comprehensive investigation of chromosome evolution and potential pathogenicity of mini-chromosomes in the Colletotrichum genus.

Benzothiazole-An Antifungal Compound Derived from Medicinal Mushroom Ganoderma lucidum against Mango Anthracnose Pathogen Colletotrichum gloeosporioides (Penz and (Sacc.)).

The present investigation is focused on exploring the possibilities of identifying biomolecules from the fruiting body of the medicinal mushroom Ganoderma lucidum against the mango anthracnose pathogen Colletotrichum gloeosporioides. The fruiting body (cap and stipe portion) of G. lucidum extracted with ethyl acetate solvent at a maximum inhibitory concentration of 1 percent exhibited the maximum mycelial growth inhibition of C. gloeosporioides with 70.10 percent and 40.77 percent, respectively. Furthermore, subjecting the ethyl acetate extracts from the cap portion of G. lucidum through thin layer chromatography (TLC) revealed the presence of two bands with Rf values of 0.38 and 0.35. The compounds eluted from band 1 recorded with the maximum mycelial growth inhibition of C. gloeosporioides by 53.77 percent followed by band 2 (46.33 percent) using an agar well diffusion test. Similarly, the analysis of ethyl acetate extracts from the cap portion of G. lucidum through Gas Chromatography-Mass spectroscopy (GC-MS) revealed the presence of the organoheterocyclic compound benzothiazole, as expressed in the highest peak area at 22.03 RT with the highest probability percentage (97%). Confirmation of the antifungal nature of benzothiazole was obtained by testing the standard sample of benzothiazole which showed a cent percent of inhibition on mycelial growth of C. gloeosporioides at 50 ppm minimum fungicidal concentration. Furthermore, benzothiazole caused abnormality in the mycelial structures, viz., distortion, shrinkage, clumping of mycelium, conidial malformation, and complete arrestment of conidial germination of C. gloeosporioides as observed through Scanning Electron Microscopy. The research on biomolecular extract of G. lucidum could be a novel and interesting concept for the possibility in suppression of plant pathogenic microbes in the natural field.

Application of Potentially Probiotic Fruit-Derived Lactic Acid Bacteria Loaded into Sodium Alginate Coatings to Control Anthracnose Development in Guava and Mango During Storage.

This study evaluated the efficacy of potentially probiotic fruit-derived lactic acid bacteria (LAB) strains loaded into sodium alginate (SA) coatings to control the anthracnose development in guava cv. Paluma and mango cv. Palmer caused by distinct pathogenic Colletotrichum species (C. asianum, C. fructicola, C. tropicale, C. siamense, C. karstii, and C. gloeosporioides) during 15 days of room temperature storage (25 ± 0.5 °C). The effects of the formulated coatings on physicochemical parameters indicative of overall postharvest quality of guava and mango were evaluated. The eight examined LAB strains caused strong inhibition on the mycelial growth of all target Colletotrichum species in vitro. LAB strains with the highest inhibitory effects (Levilactobacillus brevis 59, Lactiplantibacillus pentosus 129, and Limosilactobacillus fermentum 263) on the target Colletotrichum species were incorporated into SA coatings. These strains had viable counts of > 6 log CFU/mL in SA coatings during 15 days of room temperature storage. Application of coatings with SA + L. brevis 59, SA + L. pentosus 129, and SA + L. fermentum 263 delayed the development and decreased the severity of anthracnose lesions in guava and mango artificially contaminated with either of the tested Colletotrichum species. These coatings impacted positively on some physicochemical parameters indicative of postharvest quality and more prolonged storability of guava and mango. The formulated SA coatings loaded with tested fruit-derived potentially probiotic LAB strains could be innovative and effective strategies to control postharvest anthracnose and extend the storability of guava and mango.

Metagenomic nanopore sequencing versus conventional diagnosis for identification of the dieback pathogens of mango trees.

Dieback is one of the most dangerous fungal diseases affecting mango trees. In this study, nanopore metagenome sequencing of the root-soil samples and infected plant tissues was conducted to identify the fungal pathogens present. Soil analysis of the infected mango trees showed the abundance of the Dikarya subkingdom (59%) including Lasiodiplodia theobromae (15%), Alternaria alternata (6%), Ceratocystis huliohia and Colletotrichum gloeosporioides. Analysis of the infected plant tissues revealed the presence of A. alternata (34%). The data were deposited in the National Center of Biotechnology Information (PRJNA767267). In conclusion, nanopore metagenome sequencing analysis was a valuable tool to rapidly identify dieback-associated fungal pathogens.

Synergistic antibacterial activity of chitosan modified by double antibacterial agents as coating material for fruits preservation.

Chitosan (CS) is a natural marine polysaccharide with good biocompatibility and biodegradability. But its poor water solubility and antibacterial activity limit its application in fruits preservation. In this study, based on the advantage of quaternary phosphonium salt (QP) and salicylic acid (SA) with good antibacterial activities and different antibacterial mechanisms, a novel antibacterial coating material was synthesized by grafting QP and SA onto CS. With the grafting of SA and QP onto CS, not only the crystallinity of CS molecules decreased and the water solubility improved, but also the antibacterial activity of CS-QP-SA against Escherichia coli and Staphylococcus aureus, and Colletotrichum gloeosporioides (anthracnose) improved by the synergistic effect of QP and SA. After 20 days storage, the mango fruits treated by CS-QP-SA had a weight loss rate of 12.86 %, the fruit decay incidence was 52.00 ± 1.70 %. Hence, the CS-QP-SA films effectively extending the storage time of mango fruits to a certain extent. The results of this study indicated that CS-QP-SA might be a promising preservative for fruits and vegetables.

Antifungal activity and molecular mechanisms of mulberrin derivatives against Colletotrichum gloeosporioides for mango storage.

In this work, by using high throughput virtual screening and bioactivity assays, this work revealed that three natural compounds, mulberrin (Mul) exhibiting the highest anti-CYP51 activity, isoxanthohumol and (s)-isopsoralen markedly inhibited 14α-demethylase (a pivotal biosynthetic enzyme involved in the biosynthesis of ergosterol) in Colletotrichum gloeosporioides. Results of computational biology analysis demonstrated that, among the three inhibitors bound to the catalytic pocket of CYP51, Mul showed a closer distance with heme in CYP51 and a stronger binding free energy with CYP51. In vitro tests, Mul demonstrated excellent anti-Colletotrichum gloeosporioides activity by inhibiting CYP51 activity. Notably, Mul treatment decreased the bioactivity of CYP51, thereby increasing cell membrane permeability and cell death. Moreover, Mul treatment significantly prolonged the preservation period of fruits. These results suggest that Mul suppresses anthracnose in postharvest mango by inhibiting the growth of Colletotrichum gloeosporioides and can be used as a potential natural preserving agent.

Genetic diversity of Prosopis juliflora in the state of Qatar and its valuable use against postharvest pathogen of mango fruits.

Mango (Mangifera indica) is the second most internationally traded tropical fruit in the world. The fruit has high nutritional value. Its susceptibility to postharvest diseases and chill injuries increases its storage cost and put stress on exploring natural products that can increase its shelf-life. Our team has previously described Prosopis juliflora water-soluble leaf ethanolic (PJ-WS-LE) extract with fungicidal effectiveness against spoiling fungi. The present study explores P. juliflora genetic diversity in the state of Qatar and the antifungal effectiveness of the leaf extract of plants collected from different locations. The study also evaluates PJ-WS-LE extract efficacy against Alternaria. alternata and Colletotrichum gloeosporioides inoculated in mango samples and the power of the extract as coating material. P. juliflora samples collected from six different locations showed genetic and antimicrobial effectiveness similarities. They showed also similarity to the sequence representing P. juliflora 18S ribosomal RNA partial sequence, accession number JX139107.1 originated from India. PJ-WS-LE extract (8 mg/ml) has 80% efficacy in controlling A. alternata in mango and it lowers C. gloeosporioides disease severity by 53.4%. PJ-WS-LE extract (8 mg/ml) embedded in 1% chitosan maintained mango quality for 5 weeks. In vivo results of PJ-WS-LE extract highlights the potentials of the extract as chemical fungicides replacement.

Subcellular damages of Colletotrichum asianum and inhibition of mango anthracnose by dielectric barrier discharge plasma.

Colletotrichum asianum (C. asianum) is a new pathogenic fungus that causes mango anthracnose. Cold plasma is a novel non-thermal decontamination technology, which has been proven to be effective in controlling postharvest fungus. Herein, dielectric barrier discharge (DBD) plasma was used to treat C. asianum spores in sterile phosphate-buffered saline, the damages in subcellular structures of C. asianum and inhibition of mango anthracnose were evaluated. Results showed that after 9 min treatment, the spore germination rate and spore viability were decreased by 95.48% and 98.82%, respectively, and the subcellular structures were damaged (P < 0.05), leading to spores death. Besides, DBD plasma treatments could control mango anthracnose and maintain mango quality, and the disease incidence and lesion diameter of mango treated for 9 min were decreased by 48.00% and 62.95%, respectively. Therefore DBD plasma inactivated C. asianum spore, providing an alternative technique for preventing and controlling mango anthracnose.

Chitosan Sensitivity of Fungi Isolated from Mango (Mangifera indica L.) with Anthracnose.

In Mexico, the mango crop is affected by anthracnose caused by Colletotrichum species. In the search for environmentally friendly fungicides, chitosan has shown antifungal activity. Therefore, fungal isolates were obtained from plant tissue with anthracnose symptoms from the state of Guerrero in Mexico and identified with the ITS and ß-Tub2 genetic markers. Isolates of the Colletotrichum gloeosporioides complex were again identified with the markers ITS, Act, ß-Tub2, GADPH, CHS-1, CaM, and ApMat. Commercial chitosan (Aldrich, lot # STBF3282V) was characterized, and its antifungal activity was evaluated on the radial growth of the fungal isolates. The isolated anthracnose-causing species were C. chrysophilum, C. fructicola, C. siamense, and C. musae. Other fungi found were Alternaria sp., Alternaria tenuissima, Fusarium sp., Pestalotiopsis sp., Curvularia lunata, Diaporthe pseudomangiferae, and Epicoccum nigrum. Chitosan showed 78% deacetylation degree and a molecular weight of 32 kDa. Most of the Colletotrichum species and the other identified fungi were susceptible to 1 g L-1 chitosan. However, two C. fructicola isolates were less susceptible to chitosan. Although chitosan has antifungal activity, the interactions between species of the Colletotrichum gloeosporioides complex and their effect on chitosan susceptibility should be studied based on genomic changes with molecular evidence.

Formulation of the biological control yeast Meyerozyma caribbica by electrospraying process: effect on postharvest control of anthracnose in mango (Mangifera indica L.) and papaya (Carica papaya L.).

BACKGROUND: Microorganism for biological control of fruit diseases is an eco-friendly alternative to the use of chemical fungicides. RESULTS: This is the first study evaluating the electrospraying process to encapsulate the biocontrol yeast Meyerozyma caribbica. The effect of encapsulating material [Wey protein concentrate (WPC), Fibersol® and Trehalose], its concentration and storage temperature on the cell viability of M. caribbica, and in vitro and in vivo control of Colletotrichum gloeosporioides was evaluated. The processing with commercial resistant maltodextrin (Fibersol®) 30% (w/v) as encapsulating material showed the highest initial cell viability (95.97 ± 1.01%). The storage at 4 ± 1 °C showed lower losses of viability compared to 25 ± 1 °C. Finally, the encapsulated yeast with Fibersol 30% w/v showed inhibitory activity against anthracnose in the in vitro and in vivo tests, similar to yeast fresh cells. CONCLUSION: Electrospraying was a highly efficient process due to the high cell viability, and consequently, a low quantity of capsules is required for the postharvest treatment of fruits. Additionally, the yeast retained its antagonistic power during storage. © 2021 Society of Chemical Industry.

Growth behavior of low populations of Listeria monocytogenes on fresh-cut mango, melon and papaya under different storage temperatures.

The growth behavior of Listeria monocytogenes low population (1-4 cells/sample) on fresh-cut mango, melon, papaya and fruit mix stored at 4, 8, 12 and 16 °C was evaluated over 10 days. Mango showed the lowest counts for L. monocytogenes during 10 days regardless of storage temperature (<1.7 log cfu.g-1). Melon supported high bacterial growth over 10 days, reaching 5 log cfu.g-1 at 16 °C. Both the fruit and storage temperature influenced the Listeria low population growth potential (δ). Cumulative frequency distribution of L. monocytogenes showed that after 10 days, 100% of fresh-cut fruits and fruit mix stored at 4 °C remained ≤2 log cfu.g-1, while at 12 and 16 °C 100% of melon, papaya and fruit mix samples exceeded this limit. At 8 °C, 100% of mango and fruit mix samples remained below this limit after 10 days, whereas 100% of melon and papaya reached it after 7 days. Results indicate 4 °C as the ideal to store safely fresh-cut mango, melon, papaya and fruit mix for 10 days. Besides, 8 °C can also be an option, but not for melon and papaya. Findings highlight the ability of L. monocytogenes to survive and grow in fresh-cut fruits even at a very low initial population levels.

Bacterial structure and dynamics in mango (Mangifera indica) orchards after long term organic and conventional treatments under subtropical ecosystem.

This study explores the comparative effect of conventional and organic treatments on the rhizosphere microbiome of Mangifera indica cv. Dashehari. The long-term exposures (about 20 years) were monitored under a subtropical ecosystem. Based on plant growth properties and acetylene reduction assay, 12 bacterial isolates (7 from G1-organic and 5 from G2-conventional systems) were identified as Pseudomonas and Bacillus spp. In the conventional system, dehydrogenase activity significantly decreased (0.053 µg TPF formed g-1 of soil h-1) and adversely affected the bacterial diversity composition. In comparison, organic treatments had a good impact on dehydrogenase activity (0.784 µg TPF formed g-1 of soil h-1), alkaline phosphatase (139.25 µg PNP g-1 soil h-1), and bacterial community composition. The Metagenomics approach targeted the V3 and V4 regions to see the impact in the phylum, order, family, genus, and species for both the treatments. Results showed that phylum Acidobacteria (13.6%), Firmicutes (4.84%), and Chloroflexi (2.56%) were dominating in the G2 system whereas phylum Bacteroides (14.55%), Actinobacteria (7.45%), and Proteobacteria (10.82%) were abundantly dominated in the G1 system. Metagenome sequences are at the NCBI-GenBank sequence read archive with SRX8289747 (G1) and SRX8289748 (G2) in the study PRJNA631113. Results indicated that conventional and organic conditions affect rhizosphere microbiome and their environment.

Kinetic study of selected microorganisms and quality attributes during cold storage of mango and passion fruit smoothie subjected to dimethyl dicarbonate.

The objectives of this research were to study the effect of DMDC (0-250 ppm) on quality and shelf life of mango and passion fruit smoothie during cold storage. The correlation between microbial population (total microorganisms, yeast and mold, E. coli and S. aureus) and DMDC concentration using zero-order kinetic and first-order kinetic was also determined. In addition, the effect of DMDC compared with pasteurization (90 °C, 100 s) on quality of mixed mango and passion fruit smoothie during the cold storage (4 °C) was studied. The results showed that microbial inactivation was best-described by first-order kinetic model due to a higher coefficient of determination (R2). In addition, DMDC did not affect the decreasing trend of total soluble solid, color difference (∆E*) and total phenolic compound as compared to control during the cold storage. DMDC also hindered the increasing trend in microbial population and prevented the loss of antioxidant activity (DPPH and FRAP assays) and total flavonoid content and decreased the PPO activity as compared with the control during the cold storage. In summary, DMDC showed the potential to maintain the quality and to extend the shelf life of mango and passion fruit smoothie during cold storage.

Antifungal Effect of Chitosan/Nano-TiO2 Composite Coatings against Colletotrichum gloeosporioides, Cladosporium oxysporum and Penicillium steckii.

Postharvest pathogens such as C. gloeosporioides (MA), C.oxysporum (ME) and P. steckii (MF) are the causal agents of disease in mangoes. This paper presents an in vitro investigation into the antifungal effect of a chitosan (CTS)/nano-titanium dioxide (TiO2) composite coating against MA, ME and MF. The results indicated that, the rates of MA, ME and MF mortality following the single chitosan treatment were 63.3%, 84.8% and 43.5%, respectively, while the rates of mycelial inhibition were 84.0%, 100% and 25.8%, respectively. However, following the addition of 0.5% nano-TiO2 into the CTS, both the mortality and mycelial inhibition rates for MA and ME reached 100%, and the mortality and mycelial inhibition rate for MF also increased significantly, reaching 75.4% and 57.3%, respectively. In the MA, the dry weight of mycelia after the CTS/0.5% nano-TiO2 treatment decreased by 36.3% in comparison with the untreated group, while the conductivity value was about 1.7 times that of the untreated group, and the protein dissolution rate and extravasation degree of nucleic acids also increased significantly. Thus, this research revealed the potential of CTS/nano-TiO2 composite coatings in the development of new antimicrobial materials.

Resistance mechanisms and fitness of pyraclostrobin-resistant isolates of Lasiodiplodia theobromae from mango orchards.

BACKGROUND: Stem-end rot, caused by Lasiodiplodia theobromae (Pat.) Griffon & Maubl is a serious postharvest disease in mango. In China, a high prevalence of the QoI fungicides resistance has been reported in the last decade. The study aimed to discuss factors determining rapid development of pyraclostrobin-resistance and its resistance mechanisms. METHODS: To determine the resistance stability and fitness of pyraclostrobin resistance in L. theobromae, three phenotypes of pyraclostrobin resistance were compared and analyzed for the EC50 values, mycelial growth, virulence and temperature sensitivity and osmotic stress sensitivity. The relative conductivity and enzyme activities of different phenotypes were compared under fungicide stress to explore possible biochemical mechanisms of pyraclostrobin resistance in L. theobromae. The Cytb gene sequences of different phenotypes were analysed. RESULTS: All isolates retained their original resistance phenotypes during the 10 subcultures on a fungicide-free PDA, factor of sensitivity change (FSC) was approximately equal to 1. The resistance-pyraclostrobin of the field isolates should be relatively stable. Two pyraclostrobin-resistant phenotypes shared similar mycelial growth, virulence and temperature sensitivity with pyraclostrobin-sensitive phenotype. After treated by pyraclostrobin, the relative conductivity of the sensitive phenotype was significantly increased. The time of Pyr-R and Pyr-HR reached the most conductivity was about 8-10 times than that of Pyr-S, the time for the maximum value appearance showed significant differences between sensitive and resistant phenotypes. The activities of Glutathione S-transferase (GST), catalase (CAT) and peroxidase (POD) of Pyr-HR were 1.78, 5.45 and 1.65 times respectively, significantly higher than that of Pyr-S after treated by 200 mg/l pyraclostrobin. CONCLUSION: The results showed that the pyraclostrobin-resistant phenotypes displayed high fitness and high-risk. The nucleotide sequences were identical among all pyraclostrobin-resistant and -sensitive isolates. The pyraclostrobin resistance was not attributable to Cytb gene alterations, there may be some of other resistance mechanisms. Differential response of enzyme activity and cell membrane permeability were observed in resistant- and sensitive-isolates suggesting a mechanism of metabolic resistance.