Vietnam Journal of Biotechnology

Plant endophytes are an interesting group of microorganisms that colonize the internal tissues of living plants and do not cause any disease symptoms in the host plant. It exists in different parts of plants, such as roots, leaves, and stems, and significantly affects the formation of metabolic products in plants by promoting the accumulation of important secondary metabolites. The present study focused on analyzing the diversity and distribution of endophytic fungi related to different tissues from samples of the medicinal plant Dysosma difformis (Hemsl & E.H. Wilson) T.H. Wang collected in Ha Giang and Lai Chau, in which the isolates from roots were 27 strains (50.94%), 12 strains from stems (22.64%), and 14 strains from leaves (26.42%). Based on the isolates, we analyzed the fungal diversity through seven different diversity indices. The results showed that isolates’ diversity was similar to the endophytic fungal population in tissues of D. difformis distributed in different vegetation in Vietnam. Specifically, Shannon's index showed the highest diversity in roots (H′ = 2.673), followed by stems (H′ = 2.162) and leaves (H′ = 2,054). Similarly, species richness was highest in roots (Dmg = 4,551; Dmn = 3,079) and stem (Dmg = 4,024; Dmn = 3,175) and lowest in leaves (Dmg = 3.41; Dmn = 2,405). However, the Simpson diversity index showed that the endophytic fungal community was most abundance in leaves (1-D = 0.911), followed by stems (1-D = 0.897) and roots (1-D = 0.867). In addition, the Sorensen index of 0.615 shows the average similarity in species composition between the two sites, Ha Giang and Lai Chau. This is the first report on the diversity of endophytic fungi isolated from D. difformis, paving a potential way for screening endogenous fungal strains capable of producing important secondary compounds.

Mục lục

Five entomopathogenic nematode strains, included 3 strains of the genus Steinernema, e.g. S-PQ16, SS-CP12 and S-TX1, and two strains of genus Heterorhabditis indica, e.g. H-CB3452 and H-KT3987 were evaluated on virulence and reproduction capacity on white grubs of black scarab (Alissonotum impressicolle Arrow) a serious pest damaging in the soils of sugarcane and many economic crops in Western Highland, particular Lam Dong province. The experiments were evaluated the pathogenicity virulence of EPN strains through establishing index as lethal concentration of 50 mortality percentage of host insects (LC50). While the reproduction capacity of EPN strains were established yield of infective juveniles (IJs) that produced inside insect cadavers. The bioassays on virulence of S-PQ16, S-CP12, S-TX1, H-CB3452 and H-KT3987 were revealed the mortality of white grubs as 93.3%, 86.7%; 93.3%; 86.7% and 73.3%, respectively, at the highest concentration of 5,000 IJs/insect. The 50% mortality of five indigenous strains was high levels with LC50 values ranged between 1,362 and 2,725 IJs. These values are also similar with our results previously on white grubs with some other EPN indigenous strains. It is also suitable with some evaluation bioassays of EPN on white grubs reported from China and Australia. The IJs yields from the insect cadavers were obtained up to 31×103 IJs with the strain S- CP12; 59.7×103 IJs with the S-PQ16 strain and 73.5×103 IJs with the strain S-TX1. Particularly, the highest yields were obtained from two strains of Heterorhabditis indica, viz. 125.1×103 IJs with H-KT3987 strain and 112.6×103 IJs with H-CB3452 strains. Respect to high virulence and also high reproduction capacity all these EPN strains should be satisfied the biological agents that can be used for biological control of white grubs of pests are living in the soil environment.

Arbuscular mycorrhizal (AM) fungi are soil eukaryotes that belong to phylum Glomeromycota and have symbiosis with the vast majority of higher plants’ roots. AM fungi are believed to be coevolved with terrestrial plants, the abundance and diversity of AM fungal communities as a result are host plant dependent. A survey of AM fungi from the rhizospheres of medicinal plants in Northern Vietnam including gurma Gymnema sylvestre and turmeric Curcuma longa was carried out. From the extracted total DNAs of the medicinal plants’ rhizosphere soil samples, 35 mycorrhizal fungal species were identified by analyzing small subunit rRNA gene sequences. Result revealed that genus Glomus is the most abundant in the AM communities of G. sylvestre and C. longa, followed by Gigaspora and Acaulospora. Besides, AM species belonging to genera Scutellospora, Diversispora and Rhizophagus were observed in almost all rhizosphere soil samples. The spore counting by wet sieving and decanting method uncovered a variation in AM spore density of gurma and turmeric rhizosphere. In general, AM species were found more abundantly and more diverse in collected rhizome soil samples of C. longa (27 species belonging to 10 genera) than of G. sylvestre (17 species found belonging to 7 genera). The observed difference in AM communities of G. sylvestre and C. longa supports evidence for the dependence of AM fungal species on host plants, and indicates that AM fungi may have relation to the host plants’ secondary metabolite production.

Biosurfactants are amphiphilic molecules with effective surface-active and biological properties applicable to replace synthetic surfactant in petroleum industry. Interest in microbial surfactants has been steadily increasing in recent years, as they have numerous advantages compared to chemical surfactants including a lower toxicity, better environmental compatibility and effective properties at extreme temperature, pH levels and salinity. A crude oil-degrading yeast strain (C. 1214-BK14) was selected among the isolated strains as a potential biosurfactant-producer from producing oil wells at White Tiger oilfield because of its ability to produce biosurfactant using crude oil as a sole carbon source. An emulsification index (E24%) of 57% was obtained initially by Candida tropicalis 1214-BK14 in previous study. Therefore, the optimization of biosurfactant production of this strain for enhanced crude oil degradation was carried out based on central composite design and analyzed using response surface methodology (RSM). The biosurfactant production process was investigated as function of three independent variables: crude oil (2.5-5 % w/v), (NH4)2SO4 (0.35-0.45% w/v), and solution pH (5-8). RSM analysis showed that the optimum condition for the biosurfactant production by C. tropicalis 1214-BK14 were 6.1, 3.97% (w/v) and 0.37% (w/v) for pH, concentration of carbon (crude oil) and nitrogen substrate ((NH4)2SO4), respectively, with the emulsification index measured in the conditions was 80.2%. The total crude oil and C10-C43 alkanes degradation efficiency by this strain estimated using GC/MS were 89.8% and 80.47-98.58%, respectively. These results revealed that the strain Candida tropicalis 1214-BK14 exhibited a tremendous potential for contaminated-crude oil degradation and microbial enhanced oil recovery (MEOR).

Opisthorchiasis is a zoonotic parasitic infection caused by small liver fluke species, Opisthorchis viverrini,O. felineus and Clonorchis sinensis, in the family Opisthorchiidae. Vietnam has both species, of which C.sinensis is distributed in the northern and O. viverrini in the central provinces. In addition to the mitochondrialgenomes, the ribosomal DNA sequences (rDNA) of these species are highly needed to obtain for providingmolecular markers in species identification, classification, phylogeny and evolutionary studies. In this study,the near/complete nucleotide sequences of ribosomal transcription units (rTU) from O. viverrini (Vietnamesesample), O. felineus (Russian sample) and C. sinensis (Vietnamese sample) were analyzed. All rTUs for threespecies were determined, which is 7,839 bp for O. viverrini, 6,948 bp for O. felineus and 7,296 bp for C.sinensis containing structures of 18S, ITS1, 5,8S, ITS2 and 28S. The IGS region was not obtained for all threespecies. In all three species, sequence analysis revealed 2 tandem repetitive elements of 47-48 bp/each in ITS1but not in ITS2. The nucleotide sequences of 18S, ITS1, ITS2 and 28S are valuable ribosomal markers that thisstudy provides for diagnosis, identification, taxonomic classification and population genetics. In conclusion,the rTU sequences for the three species of the family Opisthorchiidae have been identified and providesmolecular markers for the use of phylogenetic analysis for species/family classification in the superfamilyOpisthorchioidea and the class Trematoda.

Hypercholesterolemia is a major cause of cholesterol build-up in the coronary arteries, which can subsequently lead to heart disease or atherosclerosis. Cholesterol levels can be lowered by cholesterol-lowering drugs but some of these drugs may have harmful side effects, while supplementation of Lactobacillus has shown the potential to reduce serum cholesterol levels by virtue of bile salt hydrolase (bsh) activity. In this study, Lactobacillus plantarum VFE-04, L. rhamnosus VFE-08, and L. plantarum VFE-14, had been isolated from Vietnamese healthy adults, were able to deconjugate glycodeoxycholate (GDC) on MRS plates and MRS broth supplemented with GDC. In addition, deconjugating activity of L. plantarum VFE-04, L. rhamnosus VFE-08, and L. plantarum VFE-14 were also found in cell-free extract as expressed by amount of glycine that released in the supernatant. Four bsh genes including bsh1, bsh2, bsh3, and bsh4 have been identified by PCR in these strains. In addition, L. plantarum VFE-04, L. rhamnosus VFE-08, and L. plantarum VFE-14 also showed high ability to resist bile salts and low pH. The results of 16S rRNA gene analyses showed that L. plantarum VFE-04, L. rhamnosus VFE-08, and L. plantarum VFE-14 and had high similarity scores with L. plantarum ZZU 23 (100%), L. rhamnosus JCM 1136 (99%) and L. plantarum S7 (98.65%), respectively. This study suggests that L. plantarum VFE-04, L. rhamnosus VFE-08, and L. plantarum VFE-14 have the potential to be explored as probiotics in the management of hypercholesterolaemia in near future.

Streptococcus suis (S. suis) là một trong những tác nhân gây bệnh quan trọng nhất trong viêm màng não mủ cấp trên người lớn tại Việt Nam và một số nước châu Á như Thái Lan, Hong Kong. Chúng tôi khảo sát khả năng phát triển phương pháp ELISA chẩn đoán đặc hiệu sử dụng protein bề mặt của S. suis SAO làm kháng nguyên. Kháng thể kháng protein SAO được sản xuất trên mô hình thỏ nhằm mục đích sử dụng trong xây dựng qui trình ELISA kiểu mẫu. S. suis và các tác nhân vi trùng khác được dùng trong phản ứng ELISA để xác định độ đặc hiệu và độ nhạy của qui trình. Nhằm đánh giá khả năng ứng dụng trong khảo sát huyết thanh học trên người, qui trình ELISA được đánh giá hiệu quả với mẫu huyết thanh của bệnh nhân. Qui trình ELISA kiểu mẫu được xác định với nồng độ kháng nguyên SAO-M phủ giếng tối ưu là 0.1mg/giếng, nồng độ pha loãng của huyết thanh thỏ chứa kháng thể kháng SAO-M  và của kháng thể thứ cấp lần lượt là 1:500 và 1:10000. Huyết thanh thỏ chứa kháng thể kháng SAO-M có độ đặc hiệu 100%với hiệu giá kháng thể là 1:16000. Qui trình ELISA kiểu mẫu có độ đặc hiệu 95%  và độ nhạy 90% khi thử nghiệm trên huyết thanh người được xác định nhiễm với S. suis hay với các tác nhân phổ biến khác gây bệnh viêm màng não mủ tại Việt Nam. Kết quả nghiên cứu khẳng định thành công bước đầu trong việc xây dựng qui trình ELISA chẩn đoán nhiễm S. suis, và cho thấy tiềm năng ứng dụng trong tương lai.

The pathogenic fungi often cause huge impacts on agricultural crops, and occupy over 80% of plant diseases. Fusarium oxysporum and Rhizoctonia solani are fungal pathogens that can lead to rapid development of plant diseases on important crops in Tay Nguyen (e.g., pepper, coffee, rubber, cashew). Therefore, the study of microorganisms with bioactivity against these pathogens is essential to control plant diseases. In this study, we isolated microorganisms from rhizospheres of pepper in Tay Nguyen and screened beneficial microbes against two pathogenic fungi using agar well diffusion assay. Obtained results showed that there are different about isolated microbial density between samples collected from diseased and healthy pepper. The bacterial population is higher in rhizosphere region of healthy pepper than in those of diseased plants. In contrast, fungal density is lower in rhizosphere region of healthy plants than in those of diseased ones. From isolation plates, we selected and purified 391 strains including 236 bacteria, 149 actinomycetes and 6 fungi for screening antifungal activity. Out of isolated microorganisms, 44 strains (36 bacteria, 6 actinomycetes, and 2 fungi) showed antagonistic activity against at least one of two pathogens (F. oxysporum and R. solani), of which 15 isolates showed activity against both fungi. Identification of isolates with highest activity using the 16S rRNA gene sequences showed bacterial strains belonged to different species Enterobacter ludwigii, Pseudomonas fulva, Bacillus subtilis, whereas 2 actinomycetes belonged to the genus Streptomyces: Streptomyces sp. and Streptomyces diastatochromogenes. Identification of the isolated fungus based on morphological characteristics and the 18S rRNA gene sequence revealed that this strain belonged to species Penicillium oxalicum. Our study revealed the potential of the indigenous microorganisms in preventing and controlling plant-pathogenic fungi.

Mango (Mangifera indica L.) is one of the most popular and nutritious fruits cultivated widely in Vietnam. However, under increasingly harsh climate conditions, mangoes are easily susceptible to fungal invasion and spoiled, thereby reducing mango yield. Therefore, this study was carried out to determine the pathogenic fungal strains of mango to provide useful information for finding effective measures to prevent the diseases. Rotten mango fruits were collected from different markets in Hanoi, Vietnam. Three fungal strains (M1, M2 and M3) were isolated from the studied mangoes samples. All strains were demonstrated as fungal agents associated with mango rot through pathogenicity tests. Microscopic observation showed that the mycelium of these fungal strains was branched and septate. M1 strain formed dark-brown conidiophores and conidia produced on conidiophores. M2 strain produced α- and β-conidia, as well as sub-ovoid and brown sclerotia. Whereas the M3 strain could not produce spores. Additionally, this study determined that all three fungal isolates showed the fastest growth on PDA at 30oC. The optimum growth of the M1 and M3 strains was observed at pH 5.0 while the M2 strain grew actively at pH 7.0 and 8.0. All selected strains showed the ability to produce extracellular enzymes, in which the M1 strain synthesized both cellulase and pectinase while the M2 and M3 strain secreted only pectinase. Finally, by molecular identification method, the isolates (M1, M2 and M3) were identified as Aspergillus niger isolate M1, Phomopsis sp. M2, Lasiodiplodia theobromae M3, respectively.