Four New Chromones from the Endophytic Fungus Phomopsis asparagi DHS-48 Isolated from the Chinese Mangrove Plant Rhizophora mangle

Four new chromones, phomochromenones D–G (1–4), along with four known analogues, diaporchromone A (5), diaporchromanone C (6), diaporchromanone D (7), and phomochromenone C (8), were isolated from the culture of Phomopsis asparagi DHS-48 from Chinese mangrove Rhizophora mangle. Their structures were elucidated on the basis of comprehensive spectroscopic analysis. The absolute configurations of 1 and 4 were assigned on the basis of experimental and calculated electronic circular dichroism (ECD) data, and those of enantiomers 2 and 3 were determined by a modified Mosher’s method and basic hydrolysis. To the best of our knowledge, phomochromenones D–F (1–4) possessing a 3-substituted-chroman-4-one skeleton are rarely found in natural sources. Diaporchromone A (5) showed moderate to weak immunosuppressive activity against T and/or B lymphocyte cells with IC50 of 34 μM and 117 μM.

Diversity of Endophytic Fungi of Psidium guajava (Myrtaceae) and Their Antagonistic Activity against Two Banana Pathogens

Aims: The present study was carried out to determine the diversity of endophytic fungi that colonize the leaves of Psidium guajava, and to evaluate their antagonistic activity against Fusarium oxysporum f.sp. cubense and Mycosphaerella fijiensis which are the two main phytopathogens of banana plants. Place and Duration of Study: The research was carried out at Microbiology Laboratory, Faculty of Sciences, University of Yaoundé I and Microbiology Laboratory, Faculty of Biotechnologies, University of Agronomic Sciences and Veterinary Medicine Bucharest, between April 2018 and February 2020. Methodology: Fragments of surface sterilized leaves of Psidium guajava were inoculated on Potato Dextrose Agar supplemented with chloramphenicol. The isolated and purified endophytic fungi were identified based on their macroscopic and microscopic characters using a mycological atlas as guide. The non-sporulating isolates were identified by comparing the ITS regions of their DNA to those of known fungi registered in the GenBank database. The antagonistic activity of the endophytic fungi isolated against Fusarium oxysporum and Mycosphaerella fijiensis was screened using dual culture method. Results: A total of 28 endophytic fungal were isolated from the leaves of Psidium guajava corresponding to a colonization frequency of 33.33%. These isolates were identified as: Aspergillus sp., Botryosphaeria sp., Fusarium sp., Neoscytalidium sp., Xylaria sp., Phyllosticta capitalensis, Cercospora apii, Xylaria longipes, Phomopsis sp., Phomopsis asparagi, Aspergillus versicolor, Pallidocercospora thailandica, and Xylaria grammica that belonged to the Deuteromycota and Ascomycota divisions. These endophytic fungi inhibited the growth of Fusarium oxysporum f.sp. cubense and Mycosphaerella fijiensis with the percentage inhibition varying respectively from 23.25% to 73.52% and from 21.36% to 100%. The species Botryosphaeria sp., Phomopsis sp., Phomopsis asparagi, and Xylaria longipes exhibited the greatest activity. Conclusion: The leaves of Psidium guajava have a fairly varied diversity of endophytic fungi. These endophytic fungi can serve as potential biological control agents against Panama and Sigatoka diseases of banana and also would produce secondary metabolites with antifungal properties.

Biological Characteristics and Genetic Diversity of Phomopsis asparagi, Causal Agent of Asparagus Stem Blight

Asparagus stem blight is a regional disease. In the present study, we compared strains of Phomopsis asparagi from six different provinces to determine their biological characteristics and genetic diversity, differences in the pycnidium and conidium production, pathogenicity, and growth rate. Considerable differences were established in the pycnidium and conidium production among the P. asparagi strains from the six studied provinces. The largest pycnidium and conidium production had the strains from Fujian, followed by those from Hainan. The virulence of P. asparagi strains was significantly different but without a correlation with the geographical source of the strain. FJ2 had the highest virulence, followed by HN2, SD4, and SD5, whereas SD5 had the lowest virulence. The colony diameter and dry weight of the strains of asparagus stem blight fungus from the six provinces were substantially different. The colonies of HN1-5 had the largest diameters, whereas those of XT1-5, LT1-3, FJ1-5, and SX6 had smaller diameters. Four primers with good repeatability and strong specificity were selected from 100 intersimple sequence repeat (ISSR) primers. ISSR-PCR amplification was performed on 36 strains of asparagus stem blight fungus, and a large number of repeatable DNA fingerprints were obtained. Most of the amplified fragments were within 300 to 500 bp. In all, 69 total points, 64 multiple points, and 92.75% polymorphism points were established. The number of ISSR gene sites detected by four primers ranged from 14 to 20, with an average of 16 multiple sites. The copolymerization was divided into three groups: XT1-5, LT1-3, and FJ1-5, which were clustered into the first group; SD1-6, SX1-6, and HB1-6, clustered into the second group; and HN1-5 in the third group. The results of the cluster analysis revealed that the strains of the neighboring provinces had a nearer phylogenetic relationship than that between distant ones. Therefore, the system evolution of P. asparagi is related to the geographical distribution of its strains.

Can metal-tolerant endophytic biocontrol agents promote plant-growth under metal stress?

Five metal-tolerant endophytic isolates (Bipolaris sp. LF7, Diaporthe miriciae LF9, Trichoderma asperellum LF11, Phomopsis asparagi LF15, Saccharicola bicolor LF22), with known metal-tolerance attributes and biocontrol activities against Ganoderma boninense, were tested for growth-promoting activities independent of (in vitro) and associated with plants (height, weight, root mass and stem circumference) (in vivo). Results revealed that metal-tolerant endophytes did not significantly render benefit to host plants as plant growth was compromised by the presence of metals. Lower production of indole-acetic acid (0.74-21.77 μg mL-1), siderophores (8.82-90.26%), and deaminase activities of 1-aminocyclopropane carboxylic acid (3.00-69.2 μmol mg protein-1 hr-1) were observed.

Comparative Metabolome and Transcriptome Analyses of Susceptible Asparagus officinalis and Resistant Wild A. kiusianus Reveal Insights into Stem Blight Disease Resistance

Phomopsis asparagi is one of the most serious fungal pathogens, which causes stem blight disease in Asparagus officinalis (AO), adversely affecting its production worldwide. Recently, the development of novel asparagus varieties using wild Asparagus genetic resources with natural P. asparagi resistance has become a priority in Japan due to the lack of resistant commercial AO cultivars. In this study, comparative metabolome and transcriptome analyses of susceptible AO and resistant wild Asparagus kiusianus (AK) 24 and 48 h postinoculated (AOI_24 hpi, AOI_48 hpi, AKI_24 hpi and AKI_48 hpi, respectively) with P. asparagi were conducted to gain insights into metabolic and expression changes associated with AK species. Following infection, the resistant wild AK showed rapid metabolic changes with increased levels of flavonoids and steroidal saponins and decreased asparagusic acid glucose ester content, compared with the susceptible AO plants. Transcriptome data revealed a total of 21 differentially expressed genes (DEGs) as the core gene set that displayed upregulation in the resistant AK versus susceptible AO after infection with P. asparagi. Kyoto Encyclopedia of Genes and Genomes pathway analysis of these DEGs identified 11 significantly enriched pathways, including flavonoid biosynthesis and primary metabolite metabolism, in addition to plant signaling and defense-related pathways. In addition, comparative single-nucleotide polymorphism and Indel distributions in susceptible AO and resistant AK plants were evaluated using the latest AO reference genome Aspof.V1. The data generated in this study are important resources for advancing Asparagus breeding programs and for investigations of genetic linkage mapping, phylogenetic diversity and plant defense-related genes.

Evaluating the Sensitivities and Efficacies of Fungicides with Different Modes of Action Against Phomopsis asparagi

Asparagus stem blight caused by Phomopsis asparagi is a major hindrance to asparagus production worldwide. Currently, fungicides are used to manage the disease in commercial production, but resistance to common fungicides has emerged in the wild population. In the present study, 132 isolates of P. asparagi collected from different provinces in China were tested for sensitivities to pyraclostrobin, tebuconazole, and fluazinam. We also determined the efficacies of six fungicides against P. asparagi. The frequency distributions of EC50 values of the isolates tested were unimodal, but the curves for pyraclostrobin and tebuconazole had long right-hand tails. The mean EC50 values for pyraclostrobin, tebuconazole, and fluazinam were 0.0426 ± 0.0029, 0.6041 ± 0.0416, and 0.0314 ± 0.0013 μg/ml, respectively. In addition, the EC50 values for pyraclostrobin were very similar with or without salicylhydroxamic acid (SHAM), 20 μg/ml, indicating that SHAM is not needed to determine the sensitivity of P. asparagi to pyraclostrobin when using the mycelial growth inhibition assay. In greenhouse assays, Merivon (42.4% fluxapyroxad plus pyraclostrobin SC), Frown-cide (500 g/liter fluazinam SC), Cabrio (250 g/liter pyraclostrobin EC), and Nativo (75% trifloxystrobin plus tebuconazole WG) showed excellent preventive efficacy against P. asparagi. And these fungicides were more effective before inoculation than when they were applied after inoculation (P < 0.05). Therefore, these fungicides should be applied prior to infection to control stem blight. In field trials, Frown-cide, Merivon, Nativo, and Cabrio also performed good control effects, ranging from 75.2 to 86.0% in 2017 and 75.4 to 87.1% in 2018. We demonstrated that Frown-cide, Merivon, Nativo, and Cabrio had considerable potential to manage asparagus stem blight. In addition, rotations of these fungicides are essential for precluding or delaying the development of resistance and for controlling the disease.