Diversity of endophytic fungi isolated from different plant parts of Acacia mangium, and antagonistic activity against Ceratocystis fimbriata, a causal agent of Ceratocystis wilt disease of A. mangium in Malaysia.

Acacia mangium is an important wood for commercial products especially pulp and medium-density fibreboard. However, it is susceptible to Ceratocystis fimbriata infection, leading to Ceratocystis wilt. Therefore, the present work aimed to (i) establish the diversity of endophytic fungi in different plant parts of A. mangium,and (ii) evaluate the antifungal potentials of the isolated and identified endophytic fungi against C. fimbriata. Endophytic fungal identification was conducted by PCR amplification and sequencing of the internal transcribed spacer 1 (ITS1) and ITS4 regions of nuclear ribosomal DNA. A total of 66 endophytic fungi were successfully isolated from different parts of A. mangium; leaf (21), stem (13), petiole (12), root (9), flower (6), and fruit (5). The endophytic fungal isolates belonged to Ascomycota (95.5%) and Zygomycota (4.5%). For Ascomycota 13 genera were identified: Trichoderma (28.6%), Nigrospora (28.6%), Pestalotiopsis (12.7%), Lasiodiplodia (9.5%), Aspergillus (6.3%), Sordariomycetes (3%), and Neopestalotiopsis, Pseudopestalotiopsis, Eutiarosporella, Curvularia, Fusarium, Penicillium, and Hypoxylon each with a single isolate. For Zygomycota, only Blakeslea sp. (5%) was isolated. Against C. fimbriata, Trichoderma koningiopsis (AC 1S) from stem, Nigrospora oryzae (AC 7L) from leaf, Nigrospora sphaerica (AC 3F) from the flower, Lasiodiplodia sp. (AC 2 U) from fruit, Nigrospora sphaerica (AC 4P) from petiole, and Trichoderma sp. (AC 9R) from root exhibited strong inhibition for C. fimbriata between 58.33 to 69.23%. Thus, it can be concluded that certain endophytic fungi of A. mangium have the potential to be harnessed as anti-Ceratocystis agent in future biotechnological applications.

Diversity and Characterization of Endophytic Fungi Isolated From the Tropical Mangrove Species, Rhizophora mucronata, and Identification of Potential Antagonists Against the Soil-Borne Fungus, Fusarium solani.

Rhizophora mucronata is an important ecosystem entity of the Malaysian mangrove forest. Since the species grows in a harsh environment, any organism that is isolated from this species would be of huge interest due to its potential in having novel bioactive compounds. In the present work, we isolated, identified and characterized, a total of 78 fungal isolates harboring inside the leaf tissues of R. mucronata. Molecular identification using the nuclear ribosomal DNA internal transcribe spacer (ITS) sequences returned with high similarity matches to known sequences in the GenBank. Maximum likelihood analysis revealed the phylogenetic relationship of all isolates from this study. Most of the dominating fungal endophytes were from the genera Pestalotiopsis, followed by Alternaria and Cladosporium. Six isolates representing the genera Alternaria, Fusarium, Nigrospora, Pestalotiopsis, Phoma, and Xylaria, were further screened for their antagonism activities. Dual culture test assay revealed their inhibition percentages against the phytopathogenic fungus Fusarium solani between 45-66%, and 0.8-23% when using non-volatile test assay. Of the six isolates, only Fusarium lateritium and Xylaria sp. showed antibacterial activities against the pathogenic bacteria, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus, with the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) ranging from 0.5 to 2 mg/mL. The DPPH radical scavenging assay recorded a high level of antioxidant activity in Xylaria sp., 3-fold above that of F. lateritium. We demonstrate for the first time, two members belonging to the endophytic fungal community in the tropical mangrove species that have potential use as antagonists and antibacterial agents for future biotechnological applications.

Functional Analysis of Mating Type Genes and Transcriptome Analysis during Fruiting Body Development of Botrytis cinerea.

Botrytis cinerea is a plant-pathogenic fungus producing apothecia as sexual fruiting bodies. To study the function of mating type (MAT) genes, single-gene deletion mutants were generated in both genes of the MAT1-1 locus and both genes of the MAT1-2 locus. Deletion mutants in two MAT genes were entirely sterile, while mutants in the other two MAT genes were able to develop stipes but never formed an apothecial disk. Little was known about the reprogramming of gene expression during apothecium development. We analyzed transcriptomes of sclerotia, three stages of apothecium development (primordia, stipes, and apothecial disks), and ascospores by RNA sequencing. Ten secondary metabolite gene clusters were upregulated at the onset of sexual development and downregulated in ascospores released from apothecia. Notably, more than 3,900 genes were differentially expressed in ascospores compared to mature apothecial disks. Among the genes that were upregulated in ascospores were numerous genes encoding virulence factors, which reveals that ascospores are transcriptionally primed for infection prior to their arrival on a host plant. Strikingly, the massive transcriptional changes at the initiation and completion of the sexual cycle often affected clusters of genes, rather than randomly dispersed genes. Thirty-five clusters of genes were jointly upregulated during the onset of sexual reproduction, while 99 clusters of genes (comprising >900 genes) were jointly downregulated in ascospores. These transcriptional changes coincided with changes in expression of genes encoding enzymes participating in chromatin organization, hinting at the occurrence of massive epigenetic regulation of gene expression during sexual reproduction.IMPORTANCE Fungal fruiting bodies are formed by sexual reproduction. We studied the development of fruiting bodies ("apothecia") of the ubiquitous plant-pathogenic ascomycete Botrytis cinerea The role of mating type genes in apothecium development was investigated by targeted mutation. Two genes are essential for the initiation of sexual development; mutants in these genes are sterile. Two other genes were not essential for development of stipes; however, they were essential for stipes to develop a disk and produce sexual ascospores. We examined gene expression profiles during apothecium development, as well as in ascospores sampled from apothecia. We provide the first study ever of the transcriptome of pure ascospores in a filamentous fungus. The expression of numerous genes involved in plant infection was induced in the ascospores, implying that ascospores are developmentally primed for infection before their release from apothecia.

The complete chloroplast genome of Aquilaria malaccensis Lam. (Thymelaeaceae), an important and threatened agarwood-producing tree species.

Known for its valuable agarwood, Aquilaria malaccensis Lam. is an evergreen tropical forest tree species endemic to the Indo-malesian region. Indiscriminate damaging and harvesting of the trees in the wild have resulted in it being listed in the CITES Appendix II for controlled trade and in the IUCN Red List as 'Vulnerable (VU)'. In this study, the complete chloroplast genome of A. malaccensis was assembled using data from high-throughput Illumina sequencing. The chloroplast genome was 174,832 bp in size, which included two inverted repeat regions of 42,091 bp each, separated by a large single copy region of 87,302 bp and a small single copy region of 3,348 bp. A total of 139 genes were predicted, including 39 tRNA, 8 rRNA, and 92 protein-coding genes. Phylogenetic analysis placed A. malaccensis within the family Thymelaeaceae. The chloroplast genome sequence of A. malaccensis offers a useful resource for future studies on the taxonomy and conservation of the threatened Aquilaria trees.

Analysis of Cryptic, Systemic Botrytis Infections in Symptomless Hosts.

Botrytis species are generally considered to be aggressive, necrotrophic plant pathogens. By contrast to this general perception, however, Botrytis species could frequently be isolated from the interior of multiple tissues in apparently healthy hosts of many species. Infection frequencies reached 50% of samples or more, but were commonly less, and cryptic infections were rare or absent in some plant species. Prevalence varied substantially from year to year and from tissue to tissue, but some host species routinely had high prevalence. The same genotype was found to occur throughout a host, representing mycelial spread. Botrytis cinerea and Botrytis pseudocinerea are the species that most commonly occur as cryptic infections, but phylogenetically distant isolates of Botrytis were also detected, one of which does not correspond to previously described species. Sporulation and visible damage occurred only when infected tissues were stressed, or became mature or senescent. There was no evidence of cryptic infection having a deleterious effect on growth of the host, and prevalence was probably greater in plants grown in high light conditions. Isolates from cryptic infections were often capable of causing disease (to varying extents) when spore suspensions were inoculated onto their own host as well as on distinct host species, arguing against co-adaptation between cryptic isolates and their hosts. These data collectively suggest that several Botrytis species, including the most notorious pathogenic species, exist frequently in cryptic form to an extent that has thus far largely been neglected, and do not need to cause disease on healthy hosts in order to complete their life-cycles.

Functional analysis of hydrophobin genes in sexual development of Botrytis cinerea.

Hydrophobins are small secreted fungal proteins that play roles in growth and development of filamentous fungi, i.e. in the formation of aerial structures and the attachment of hyphae to hydrophobic surfaces. In Botrytis cinerea, three hydrophobin genes have been identified. Studies by Mosbach et al. (2011) showed that hydrophobins are neither involved in conferring surface hydrophobicity to conidia and aerial hyphae of B. cinerea, nor are they required for virulence. The present study investigated the role of hydrophobins in sclerotium and apothecium development. Expression analysis revealed high expression of the Bhp1 gene during different stages of apothecium development. Two Bhp1 splice variants were detected that differ by an internal stretch of 13 amino acid residues. Seven different mutants in which either a single, two or three hydrophobin genes were knocked out, as well as two wild type strains of opposite mating types, were characterized for sclerotium and apothecium development. No aberrant morphology was observed in sclerotium development when single deletion mutants in hydrophobin genes were analyzed. Sclerotia of double knock out mutant ΔBhp1/ΔBhp3 and the triple knock out mutant, however, showed easily wettable phenotypes. For analyzing apothecium development, a reciprocal crossing scheme was setup. Morphological aberrations were observed in crosses with two hydrophobin mutants. When the double knock out mutant ΔBhp1/ΔBhp2 and the triple knock out mutant were used as the maternal parent (sclerotia), and fertilized with wild type microconidia, the resulting apothecia were swollen, dark brown in color and had a blotched surface. After initially growing upwards toward the light source, the apothecia in many cases collapsed due to loss of structural integrity. Aberrant apothecium development was not observed in the reciprocal cross, when these same mutants were used as the paternal parent (microconidia). These results indicate that the presence of hydrophobins in maternal tissue is important for normal development of apothecia of B. cinerea.

A novel Botrytis species is associated with a newly emergent foliar disease in cultivated Hemerocallis.

Foliar tissue samples of cultivated daylilies (Hemerocallis hybrids) showing the symptoms of a newly emergent foliar disease known as 'spring sickness' were investigated for associated fungi. The cause(s) of this disease remain obscure. We isolated repeatedly a fungal species which proved to be member of the genus Botrytis, based on immunological tests. DNA sequence analysis of these isolates, using several different phyogenetically informative genes, indicated that they represent a new Botrytis species, most closely related to B. elliptica (lily blight, fire blight) which is a major pathogen of cultivated Lilium. The distinction of the isolates was confirmed by morphological analysis of asexual sporulating cultures. Pathogenicity tests on Hemerocallis tissues in vitro demonstrated that this new species was able to induce lesions and rapid tissue necrosis. Based on this data, we infer that this new species, described here as B. deweyae, is likely to be an important contributor to the development of 'spring sickness' symptoms. Pathogenesis may be promoted by developmental and environmental factors that favour assault by this necrotrophic pathogen. The emergence of this disease is suggested to have been triggered by breeding-related changes in cultivated hybrids, particularly the erosion of genetic diversity. Our investigation confirms that emergent plant diseases are important and deserve close monitoring, especially in intensively in-bred plants.