Genetic diversity and antibacterial activity of endophytic fungi from Zanthoxylum nitidum / 中国中药杂志

In order to reveal the distribution and population characteristics of endophytic fungi from Zanthoxylum nitidum and the antibacterial potential,this study performed molecular identification and analyzed the genetic diversity and antibacterial activity of endophytic fungi from Z. nitidum in Guangxi. Through culture and molecular identification,35 strains,belonging to 15 genera,12 families,10 orders,4 classes,and 2 phyla,were isolated from various tissues of Z. nitidum,of which Colletotrichum and Fusarium were the dominant genera,respectively accounting for 20% of total strains. The diversity of endophytic fungi was significantly different among roots,stems,and leaves,as manifested by the significantly higher Shannon index( H') in stems( 1. 678) than in roots( 0. 882 1) and leaves( 0. 515 4). The antimicrobial activity analysis showed that 14. 28% of endophytic fungi inhibited at least one indicator pathogen. Among them,Fusarium sp. ZN-34 and Fusarium sp. ZN-26 separately demonstrated the strongest inhibitory effect on Escherichia coli and Staphylococcus aureus. In general,Fusarium sp. ZN-26 and Phialemoniopsis plurioloculosa ZN-35 were advantageous in suppressing the two bacteria owing to the broad spectrum and strong efficacy. In summary,Z. nitidum in Guangxi boasts rich endophytic fungi with the majority showing strong antibacterial activity,which can be used as candidates for the extraction and separation of basic antibacterial substances and the development of natural antibacterial agents.

Research strategies for endophytes in medicinal plants based on high-throughput sequencing and traditional culture and isolation methods / 中国中药杂志

The research on endophytes of medicinal plants mainly relies on the traditional culture and isolation methods. Because of their functions such as promoting host growth, improving stress resistance, promoting the accumulation of medicinal active ingredients or directly producing medicinal active ingredients, the endophytes of medicinal plants have gradually attracted wide attention. However, it was found that the strains isolated by traditional methods were not the true dominant endophytes of medicinal plants by comparing the results of traditional culture isolation with high-throughput sequencing. The blind and random nature of traditional methods leads to the lack of standards in terms of medium selection, culture time and interaction between species. On the contrary, high-throughput sequencing technology is an emerging molecular biology technology developed in recent decades. Due to its high resolution level and indepen-dent culture, it can be used for thorough analysis of the community structure and diversity of environmental microorganisms. Therefore, we proposed the strategy of using high-throughput sequencing technology to guide the traditional culture and isolation of endophytes from medicinal plants. Firstly, the endophytic structure and diversity of medicinal plants were completely clear by high-throughput sequencing technology, and the dominant endophytes of the host were unequivocal. Then according to the characteristics of each dominant endophytes design or query suitable medium for its growth to culture and isolation. Finally, the function of the isolates was studied. This method can prevent researchers from missing out on the important functional strains of the host, expand the research scope of endophytes of medicinal plants, and facilitate the in-depth excavation and utilization of endophytes of medicinal plants.

The potential of compounds isolated from Xylaria spp. as antifungal agents against anthracnose

ABSTRACT Anthracnose is a crop disease usually caused by fungi in the genus Colletotrichum or Gloeosporium. These are considered one of the main pathogens, causing significant economic losses, such as in peppers and guarana. The current forms of control include the use of resistant cultivars, sanitary pruning and fungicides. However, even with the use of some methods of controlling these cultures, the crops are not free of anthracnose. Additionally, excessive application of fungicides increases the resistance of pathogens to agrochemicals and cause harm to human health and the environment. In order to find natural antifungal agents against guarana anthracnose, endophytic fungi were isolated from Amazon guarana. The compounds piliformic acid and cytochalasin D were isolated by chromatographic techniques from two Xylaria spp., guided by assays with Colletotrichum gloeosporioides. The isolated compounds were identified by spectrometric techniques, as NMR and mass spectrometry. This is the first report that piliformic acid and cytochalasin D have antifungal activity against C. gloeosporioides with MIC 2.92 and 2.46 µmol mL-1 respectively. Captan and difenoconazole were included as positive controls (MIC 16.63 and 0.02 µmol mL-1, respectively). Thus, Xylaria species presented a biotechnological potential and production of different active compounds which might be promising against anthracnose disease.

Draft genome sequence of Paraburkholderia tropica Ppe8 strain, a sugarcane endophytic diazotrophic bacterium

Abstract Paraburkholderia tropica (syn Burkholderia tropica) are nitrogen-fixing bacteria commonly found in sugarcane. The Paraburkholderia tropica strain Ppe8 is part of the sugarcane inoculant consortium that has a beneficial effect on yield. Here, we report a draft genome sequence of this strain elucidating the mechanisms involved in its interaction mainly with Poaceae. A genome size of approximately 8.75 Mb containing 7844 protein coding genes distributed in 526 subsystems was de novo assembled with ABySS and annotated by RAST. Genes related to the nitrogen fixation process, the secretion systems (I, II, III, IV, and VI), and related to a variety of metabolic traits, such as metabolism of carbohydrates, amino acids, vitamins, and proteins, were detected, suggesting a broad metabolic capacity and possible adaptation to plant association.

Mangrove endophyte promotes reforestation tree (Acacia polyphylla) growth

ABSTRACT Mangroves are ecosystems located in the transition zone between land and sea that serve as a potential source of biotechnological resources. Brazil's extensive coast contains one of the largest mangrove forests in the world (encompassing an area of 25,000 km2 along all the coast). Endophytic bacteria were isolated from the following three plant species: Rhizophora mangle, Laguncularia racemosa and Avicennia nitida. A large number of these isolates, 115 in total, were evaluated for their ability to fix nitrogen and solubilize phosphorous. Bacteria that tested positive for both of these tests were examined further to determine their level of indole acetic acid production. Two strains with high indole acetic acid production were selected for use as inoculants for reforestation trees, and then the growth of the plants was evaluated under field conditions. The bacterium Pseudomonas fluorescens (strain MCR1.10) had a low phosphorus solubilization index, while this index was higher in the other strain used, Enterobacter sp. (strain MCR1.48). We used the reforestation tree Acacia polyphylla. The results indicate that inoculation with the MCR1.48 endophyte increases Acacia polyphylla shoot dry mass, demonstrating that this strain effectively promotes the plant's growth and fitness, which can be used in the seedling production of this tree. Therefore, we successfully screened the biotechnological potential of endophyte isolates from mangrove, with a focus on plant growth promotion, and selected a strain able to provide limited nutrients and hormones for in plant growth.

Screening of endophytic fungi stored in a culture collection for taxol production

Abstract In this work, four isolates of endophytic fungi (Alternaria alternata, Colletotrichum gloesporioides, Glomerella cingulata and Nigrospora sphaerica), deposited in the culture collection 'University Recife Mycologia' (URM) at the Universidade Federal de Pernambuco, were characterized for the genes ITS 1 and 4 (region 5.8 S) and evaluated for taxol production.

Endophytic Bacillus strains enhance pearl millet growth and nutrient uptake under low-P

Abstract Bacterial endophytes are considered to have a beneficial effect on host plants, improving their growth by different mechanisms. The objective of this study was to investigate the capacity of four endophytic Bacillus strains to solubilize iron phosphate (Fe-P), produce siderophores and indole-acetic acid (IAA) in vitro, and to evaluate their plant growth promotion ability in greenhouse conditions by inoculation into pearl millet cultivated in a P-deficient soils without P fertilization, with Araxá rock phosphate or soluble triple superphosphate. All strains solubilized Fe-P and three of them produced carboxylate-type siderophores and high levels of IAA in the presence of tryptophan. Positive effect of inoculation of some of these strains on shoot and root dry weight and the N P K content of plants cultivated in soil with no P fertilization might result from the synergistic combination of multiple plant growth promoting (PGP) traits. Specifically, while B1923 enhanced shoot and root dry weight and root N P content of plants cultivated with no P added, B2084 and B2088 strains showed positive performance on biomass production and accumulation of N P K in the shoot, indicating that they have higher potential to be microbial biofertilizer candidates for commercial applications in the absence of fertilization.

Diversity and communities of culturable endophytic fungi from different tree peonies (geoherbs and non-geoherbs), and their biosynthetic potential analysis

Abstract To know more about the potential roles of endophytic fungi in the formation mechanism of Daodi medicinal material, diversity and communities of culturable endophytic fungi in three types of tree peonies were investigated. Endophytic fungi of three types of tree peonies were isolated and identified. The diversity was analyzed. Bayesian trees constructed by MrBayes 3.2.6 after phylogenetic analysis of the ITS sequences. The endophytic fungi potential for synthesis of natural products was assessed by means of detecting NRPS and PKS gene sequences. In total, 364 endophytic fungi isolates representing 26 genera were recovered from Paeonia ostii 'Feng Dan', Paeonia ostii 'Luoyang Feng Dan', and Paeonia suffruticosa 'Luoyang Hong'. More culturable endophytic fungi appeared in P. suffruticosa 'Luoyang Hong' (206) compared with P. ostii 'Feng Dan' (60) and P. ostii 'Luoyang Feng Dan' (98). The fungal community of P. ostii 'Feng Dan' had the highest richness and diversity. PKSs and NRPS detection rates of endophytic fungi from P. ostii 'Feng Dan' are both the highest among the three types of tree peonies. Results indicate that endophytic fungus is an important factor of Daodi Cortex Moutan forming, and endophytic fungi in peony are related to genuineness of Cortex Moutan.

Illumina-based analysis of endophytic bacterial diversity of tree peony (Paeonia Sect. Moutan) roots and leaves

ABSTRACT Diverse communities of bacteria inhabit plant tissues and those bacteria play a crucial role for plant health and growth. Tree peony (Paeonia Sect. Moutan) is known for its excellent ornamental and medicinal values as Chinese traditional plant, but little is known about its associated bacterial community under natural conditions. To examine how endophytic bacteria in tree peony vary across tissues and cultivars, PCR-based Illumina was applied to reveal the diversity of endophytic bacteria in tree peony. A total of 149,842 sequences and 21,463 operational taxonomic units (OTUs) were obtained. The OTU abundance of roots was higher than leaves across other three cultivars except for 'Kinkaku' and 'Luoyanghong'. The community was composed of five dominant groups (Proteobacteria, Firmicutes, Bacteroidetes, Acidobacteria and Actinobacteria) in all samples. Endophytic bacteria community structures had changed in leaves and roots. Sequences of Pseudomonas and Enterobacteriaceae were prevalent in root samples, whereas Succinivibrio and Acinetobacter were the dominant genus in leaf samples. Otherwise, the distribution of each dominant genus among the 5 cultivars was either varied. These findings suggested that both plant genotype and tissues contribute to the shaping of the bacterial communities associated with tree peony.

Endophytic fungus Purpureocillium sp. A5 protect mangrove plant Kandelia candel under copper stress

Abstract Mangrove is an important ecosystem in the world. Mangrove ecosystems have a large capacity in retaining heavy metals, and now they are usually considered as sinks for heavy metals. However, the mechanism of why the soil of mangrove ecosystems can retain heavy metal is not certain. In this research, endophytic fungus Purpureocillium sp. A5 was isolated and identified from the roots of Kandelia candel. When this fungus was added, it protected the growth of K. candel under Cu stress. This can be illustrated by analyzing chlorophyll A and B, RWC and WSD to leaves of K. candel. Purpureocillium sp. A5 reduces uptake of Cu in K. candel and changes the pH characterization of soil. Furthermore, A5 increase the concentration of Cu complexes in soil, and it enhanced the concentration of carbonate-bound Cu, Mn-Fe complexes Cu and organic-bound Cu in soil. Nevertheless, a significant reduction of the Cu ion was noted among A5-treated plants. This study is significant and illustrates a promising potential use for environmental remediation of endophytes, and also may partially explain the large capacity of mangrove ecosystems in retaining heavy metals.

Endophytic fungus strain 28 isolated from Houttuynia cordata possesses wide-spectrum antifungal activity

Abstract The aim of this paper is to identify and investigate an endophytic fungus (strain 28) that was isolated from Houttuynia cordata Thunb, a famous and widely-used Traditional Chinese Medicine. Based on morphological methods and a phylogenetic analysis of ITS sequences, this strain was identified as Chaetomium globosum. An antifungal activity bioassay demonstrated that the crude ethyl acetate (EtOAc) extracts of strain 28 had a wide antifungal spectrum and strong antimicrobial activity, particularly against Exserohilum turcicum (Pass.) Leonard et Suggs, Botrytis cinerea persoon and Botrytis cinerea Pers. ex Fr. Furthermore, the fermentation conditions, extraction method and the heat stability of antifungal substances from strain 28 were also studied. The results showed that optimal antifungal activity can be obtained with the following parameters: using potato dextrose broth (PDB) as the base culture medium, fermentation for 4–8 d (initial pH: 7.5), followed by extraction with EtOAc. The extract was stable at temperatures up to 80 °C. This is the first report on the isolation of endophytic C. globosum from H. cordata to identify potential alternative biocontrol agents that could provide new opportunities for practical applications involving H. cordata.

Screening and characterization of endophytic Bacillus and Paenibacillus strains from medicinal plant Lonicera japonica for use as potential plant growth promoters

Abstract A total of 48 endophytic bacteria were isolated from surface-sterilized tissues of the medicinal plant Lonicera japonica, which is grown in eastern China; six strains were selected for further study based on their potential ability to promote plant growth in vitro (siderophore and indoleacetic acid production). The bacteria were characterized by phylogenetically analyzing their 16S rRNA gene similarity, by examining their effect on the mycelial development of pathogenic fungi, by testing their potential plant growth-promoting characteristics, and by measuring wheat growth parameters after inoculation. Results showed that the number of endophytic bacteria in L. japonica varied among different tissues, but it remained relatively stable in the same tissues from four different plantation locations. Among the three endophytic strains, strains 122 and 124 both had high siderophore production, with the latter showing the highest phosphate solubilization activity (45.6 mg/L) and aminocyclopropane-1-carboxylic acid deaminase activity (47.3 nmol/mg/h). Strain 170 had the highest indoleacetic acid (IAA) production (49.2 mg/L) and cellulase and pectinase activities. After inoculation, most of the six selected isolates showed a strong capacity to promote wheat growth. Compared with the controls, the increase in the shoot length, root length, fresh weight, dry weight, and chlorophyll content was most remarkable in wheat seedlings inoculated with strain 130. The positive correlation between enzyme (cellulose and pectinase) activity and inhibition rate on Fusarium oxysporum, the IAA production, and the root length of wheat seedlings inoculated with each tested endophytic strain was significant in regression analysis. Deformity of pathogenic fungal mycelia was observed under a microscope after the interaction with the endophytic isolates. Such deformity may be directly related to the production of hydrolytic bacterial enzymes (cellulose and pectinase). The six endophytic bacterial strains were identified to be Paenibacillus and Bacillus strains based on the results of 16S rRNA gene sequencing analysis and their physiological and biochemical characteristics. Results indicate the promising application of endophytic bacteria to the biological control of pathogenic fungi and the improvement of wheat crop growth.

Search for endophytic diazotrophs in barley seeds

Eight endophytic isolates assigned to Pseudomonas, Azospirillum, and Bacillus genera according to pheno-genotypic features were retrieved from barley seeds under selective pressure for nitrogen-fixers. Genetic relationships among related isolates were investigated through RAPD. Six isolates displayed nitrogen-fixing ability, while all could biosynthesize indolacetic acid in vitro and showed no antibiosis effects against Azospirillum brasilense Az39, a recognized PGPR.

Colonization and plant growth promoting characterization of endophytic Pseudomonas chlororaphis strain Zong1 isolated from Sophora alopecuroides root nodules

The endophytic strain Zong1 isolated from root nodules of the legume Sophora alopecuroides was characterized by conducting physiological and biochemical tests employing gfp-marking, observing their plant growth promoting characteristics (PGPC) and detecting plant growth parameters of inoculation assays under greenhouse conditions. Results showed that strain Zong1 had an effective growth at 28 ºC after placed at 4-60 ºC for 15 min, had a wide range pH tolerance of 6.0-11.0 and salt tolerance up to 5% of NaCl. Zong1 was resistant to the following antibiotics (µg/mL): Phosphonomycin (100), Penicillin (100) and Ampicillin (100). It could grow in the medium supplemented with 1.2 mmol/L Cu, 0.1% (w/v) methylene blue and 0.1-0.2% (w/v) methyl red, respectively. Zong1 is closely related to Pseudomonas chlororaphis based on analysis the sequence of 16S rRNA gene. Its expression of the gfp gene indicated that strain Zong1 may colonize in root or root nodules and verified by microscopic observation. Furthermore, co-inoculation with Zong1 and SQ1 (Mesorhizobium sp.) showed significant effects compared to single inoculation for the following PGPC parameters: siderophore production, phosphate solubilization, organic acid production, IAA production and antifungal activity in vitro. These results suggest strains P. chlororaphi Zong1 and Mesorhizobium sp. SQ1 have better synergistic or addictive effect. It was noteworthy that each growth index of co-inoculated Zong1+SQ1 in growth assays under greenhouse conditions is higher than those of single inoculation, and showed a significant difference (p < 0.05) when compared to a negative control. Therefore, as an endophyte P. chlororaphis Zong1 may play important roles as a potential plantgrowth promoting agent.

Diversity of foliar endophytic fungi from the medicinal plant Sapindus saponaria L . and their localization by scanning electron microscopy

Endophytic fungi inhabit vegetable tissues or organs, without causing them any harm. Endophytes can co-evolve with plant hosts and possess species-specific interactions. They can protect the plant from insect attacks and diseases, and are also able to produce substances of biotechnological interest. In folk medicine, the bark, roots and fruits of Sapindus saponaria is used to produce substances with anxiolytic, astringent, diuretic and expectorant properties, as well as tonics, blood depuratives and cough medicine. This study evaluated the diversity of endophytic fungi present in the leaves of S. saponaria L. and observed the colonization of host plants by endophytes, using light and scanning electron microscopy. We verified that these fungi are found in intercellular and intracellular spaces. The genera of some isolates of S. saponaria were identified mainly by sequencing of ITS region of rDNA and, when possible, also by their microscopic features, as follows: Cochliobolus, Alternaria, Curvularia, Phomopsis, Diaporthe and Phoma. Phylogenetic analysis showed the existence of genetic variability of the genera Phomopsis and Diaporthe and interspecific variation among the Curvularia, Alternaria and Phoma, belonging to family Pleosporaceae.

Evaluation of foliar fungal endophyte diversity and colonization of medicinal plant Luehea divaricata (Martius et Zuccarini)

Endophyte microorganisms are organisms that live inside plants without causing any apparent damage to their hosts. Since all plants exhibit endophyte microorganisms, it is believed that mutual association is of great importance in nature. Luehea divaricata (Martius & Zuccarini), known popularly in Brazil as agoita-cavalo, is a big-sized tree with a wide distribution in the country that possesses medicinal qualities for: dysentery, leucorrhea, rheumatism, blennorrhoea, tumors, bronchitis, and depuration. This research aims at isolating and molecularly characterizing fungi isolates from L. divaricata by sequence analysis of ITS1-5.8S-ITS2 rDNA. Further, the colonization of endophyte in the host plant by Light and Scanning Electron Microscopy will also be investigated. Whereas, genera Alternaria, Cochliobolus, Diaporthe, Epicoccum, Guignardia, Phoma, and Phomopsis, were identified; rDNA sequence analysis revealed intra-species variability among endophyte isolates of the genus Phomopsis sp. Light and Scanning Electron Microscopy techniques showed the presence of endophyte fungi inside L. divaricata leaves, inhabiting inter- and intra-cellular spaces. These types of extensive colonization and dissemination were reported throughout all the leaf parts in palisade parenchyma, esclerenchyma, spongy parenchyma, adaxial epidermis, and vascular bundle indicating colonization of endophytes in múltiple structural sub-niches in the host plant.