Chemical and molecular characterization of Phomopsis and Cytospora-like endophytes from different host plants in Brazil

Phomopsis and related taxa comprise important endophytic and plant pathogenic species, and are known for the production of a diverse array of secondary metabolites. Species concepts within this group based on morphological characters and assumed host specificity do not reflect phylogenetic affinities. Additional phenotypic characters, such as profiles of secondary metabolites, are needed for practical species recognition. We investigated 36 strains of Phomopsis spp. and Cytospora-like fungi, obtained as endophytes of different host plants in Brazil, using metabolite profiling based on HPLC-UV/liquid chromatography -mass spectrometry (LC-MS) combined with cluster analysis of the results. Strains were also subjected to phylogenetic analyses based on internal transcribed spacer (ITS) rDNA. Six chemotypes were identified. Chemotypes 1-5 contained Phomopsis strains, while Cytospora-like strains formed the chemotype 6. Strains of chemotype 1 typically produced alternariols, altenusin, altenuene, cytosporones, and dothiorelones. Alternariol and seven unknown compounds were consistently produced by strains of chemotype 2. Members of chemotypes 3-5 produced poor metabolite profiles containing few chemical markers. Cytospora-like endophytes (chemotype 6) produced a characteristic set of metabolites including cytosporones and dothiorelones. Bayesian and Maximum Parsimony (MP) trees classified strains of each chemotype into single phylogenetic lineages or closely related groups. Strains of chemotypes 1 and 2 formed a monophyletic group along with Diaporthe neotheicola. The remaining Phomopsis strains formed monophyletic (chemotype 4) or polyphyletic (chemotypes 3 and 5) lineages inside a large and well supported clade. Cytospora-like strains formed a monophyletic lineage located at an intermediary position between Diaporthe/Phomopsis and Valsa/Cytospora clades. The combined results show that the production of secondary metabolites by Phomopsis and related Diaporthales may be species-specific, giving support to the use of metabolite profiling and chemical classification for phenotypic recognition and delimitation of species.     

A new fungal isolate from Paspalum scrobiculatum, Linn. with new biologically active metabolites.

A new fungal isolate from the host plant Paspalum scrobiculatum Linn., is reported. It is characterized and designated as a separate species Phomopsis paspali (n. sp.), not only for the different morphological characters as compared with the commonly occurring Phomopsis species in India, but also in view of its distinctive feature of producing biologically active new metabolites, cytochalasans. In addition, the genus Phomopsis has not been reported on this plant anywhere in the world.     

Endophytic Phomopsis species: host range and implications for diversity estimates

Foliar endophyte assemblages of teak trees growing in dry deciduous and moist deciduous forests of Nilgiri Biosphere Reserve were compared. A species of Phomopsis dominated the endophyte assemblages of teak, irrespective of the location of the host trees. Internal transcribed spacer sequence analysis of 11 different Phomopsis isolates (ten from teak and one from Cassia fistula) showed that they fall into two groups, which are separated by a relatively long branch that is strongly supported. The results showed that this fungus is not host restricted and that it continues to survive as a saprotroph in teak leaf, possibly by exploiting senescent leaves as well as the litter. Although the endophyte assemblage of a teak tree growing about 500 km from the forests was also dominated by a Phomopsis sp., it separated into a different group based on internal transcribed spacer sequence analysis. Our results with an endophytic Phomopsis sp. reinforce the earlier conclusions reached by others for pathogenic Phomopsis sp., i.e., that this fungus is not host specific, and the species concept of Phomopsis needs to be redefined.     

(+)-Menthol and its hydroxy derivatives,novel fungal monoterpenols from the fusicoccin-producing fungi,Phomopsis amygdali F6a and Niigata 2

In our search for new fusicoccins of unique diterpene glucosides from Phomopsis amygdali, we found that a fragrant substance was formed in the early stage of fusicoccin fermentation. This fragrant constituent was isolated and identified as (+)-menthol, which is a novel fungal metabolite as the enantiomer of well-known peppermint (-)-menthol. (+)-7-Hydroxymenthol and new (+)-(6S)-hydroxymenthol were also isolated and identified as fungal metabolites. In addition, p-menthanetriol, which has been reported as the first fungal monoterpene from the fungus, was also isolated. The possible biosynthetic relationship of these metabolites is discussed.     

红树林内生真菌研究进展

红树林作为一种特殊的植物群落具有丰富的内生真菌资源,目前已分离鉴定的红树林真菌超过200种,成为海洋真菌的第二大类群,已报道的红树林内生真菌主要类群是链格孢霉(Alternaria)、曲霉(Aspergillus)、芽枝霉(Cladosporium)、炭疽菌(Colletotrichum)、镰孢霉(Fusarium)、拟青霉(Paecilomyces)、拟盘多毛孢(Pestalotiopsis)、青霉(Penicillium)、茎点霉(Phoma)、拟茎点霉(Phomopsis)、叶点霉(Phyllosticta)和木霉(Trichoderma)等.大部分红树林内生真菌具有较宽的宿主范围,极少数只有单一的宿主,不同红树林植物的内生真菌区系及优势种群有很大差异.红树林内生真菌的定殖因宿主植物不同部位、植株的年龄及季节和环境的变化明显不同.红树林内生真菌能产生多种代谢产物,具有抗菌、抗肿瘤等药用价值.红树林植物内生真菌的研究和开发具有重要意义.本文综述了红树林内生真菌的生物多样性及其分布、生物学功能和次生代谢产物等方面的研究进展.     

Phylogenetic diversity of endophytic leaf fungus isolates from the medicinal tree Trichilia elegans (Meliaceae)

Various types of organisms, mainly fungi and bacteria, live within vegetal organs and tissues, without causing damage to the plant. These microorganisms, which are called endophytes, can be useful for biological control and plant growth promotion; bioactive compounds from these organisms may have medical and pharmaceutical applications. Trichilia elegans (Meliaceae) is a native tree that grows abundantly in several regions of Brazil. Preparations using the leaves, seeds, bark, and roots of many species of the Meliaceae family have been widely used in traditional medicine, and some members of the Trichilia genus are used in Brazilian popular medicine. We assessed the diversity of endophytic fungi from two wild specimens of T. elegans, collected from a forest remnant, by sequencing ITS1-5.8S-ITS2 of rDNA of the isolates. The fungi were isolated and purified; 97 endophytic fungi were found; they were separated into 17 morpho-groups. Of the 97 endophytic fungi, four genera (Phomopsis, Diaporthe, Dothideomycete, and Cordyceps) with 11 morpho-groups were identified. Phomopsis was the most frequent genus among the identified endophytes. Phylogenetic analysis showed two major clades: Sordariomycetes, which includes three genera, Phomopsis, Diaporthe, and Cordyceps, and the clade Dothideomycetes, which was represented by the order Pleosporales.     

Differential Growth Inhibition of Diaporthe and Phomopsis Isolates by the Metabolic Activity of Five Actinomycetes

Fifty-five cultures derived from Diaporthe perithecia and Phomopsis pycnidia found on diverse host plant species collected at different times and sites in Vojvodina, Yugoslavia, showed distinguishing quantitative reactions to the fungistatic activity of five actinomycetes obtained as fortuitous laboratory contaminants coming from field material. Streptomyces albidoflavus , S. albus , S. diastaticus , Streptomyces sp., and Streptoverticillium sp. could be ranked by their growth-inhibitory potential, with S. albus showing the strongest, and Streptomyces sp. the lowest. The responses of the fungi varied depending on the tested actinomycetes, but two major groups could be distinguished: A, which encompased the isolates that were less affected by the proximity of the actinomycetes; and B, with those which exhibited high sensitivity in all the experiments. Group A was typically represented by Diaporthe arctii , Phomopsis longicolla, and the Phomopsis type-1 cultures from Xanthium italicum; group B was typically represented by Diaporthe/Phomopsis helianthi, Phomopsis type-2 cultures from X. italicum , and isolates from Lactuca serriola . The results obtained underscore the dissimilarities between D. arctii and D. helianthi , and corroborate the value of the physiological aspects of congeneric isolates in considering taxonomic problems in the coelomicete genus Phomopsis.     

Metabolites from the endophytic fungus Phomopsis sp. PSU-D15

From the endophytic fungus Phomopsis sp. PSU-D15, three metabolites named as phomoenamide (1), phomonitroester (2) and deacetylphomoxanthone B (3), were isolated together with three known compounds, dicerandrol A (4), (1S,2S,4S)-p-menthane-1,2,4-triol (5) and uridine. Their structures were elucidated by spectroscopic methods. Phomoenamide (1) exhibited moderate in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Ra.     

Specifying the introgressed regions from H. argophyllus in cultivated sunflower (Helianthus annuus L.) to mark Phomopsis resistance genes

A method based upon targetting of intro-gressed markers in a Phomopsis-resistant line (R) of cultivated sunflower, issuing from a H. argophyllus cross was used to mark the Phomopsis resistance regions. Our study was based upon 203 families derived from a cross between an inbred line susceptible to Phomopsis (S1) and the introgressed resistant line (R). Families were checked for Phomopsis resistance level in a design with replicated plots and natural infection was re-inforced by pieces of contaminated stems. Thirty four primers were employed for RAPD analysis. Out of 102 polymorphic fragments between (S1) and H. argophyllus, seven were still present in (R) suggesting that they marked introgressions of H. argophyllus into (R). The plants were scored for the presence or absence of 19 fragments obtained from five primers, and the relationships between the presence/absence of fragments in plants and Phomopsis resistance/susceptiblity in the progenies was determined by using an analysis of variance. We found that at least two introgressed regions, as well as favourable factors from sunflower, contributed to the level of Phomopsis resistance in cultivated sunflower. Received: 28 June 1996 / Accepted: 5 July 1996     

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.     

多噬伯克霍尔德氏菌WS-FJ9对林木病原菌物的拮抗作用及代谢产物的稳定性

【背景】伯克霍尔德氏菌(Burkholderia)是一类重要的植物根际促生细菌,许多菌株具有抑制植物病原菌生长和促进植物生长等功能。【目的】探究高效解磷促生细菌多噬伯克霍尔德氏菌(B. multivorans) WS-FJ9对不同林木病原菌物的抑菌作用。【方法】采用平板对峙法检测菌株WS-FJ9对5株林木病原真菌和卵菌的抑制效果;基于比色法检测经菌株WS-FJ9处理后病原菌菌丝细胞内含物的变化;使用antiSMASH 5.0在线预测网站对其次生代谢物质进行预测;通过菌丝生长抑制速率法对其无菌发酵滤液的抑菌活性和稳定性进行研究。【结果】菌株WS-FJ9对5种林木病原菌均具有不同程度的抑制作用,其中菌悬液对樟疫霉(Phytophthora cinnamomi)的抑制作用最好,抑菌带宽度为14.82±0.20mm,无菌发酵滤液对真菌拟茎点霉(Phomopsismacrospore)和松杉球壳孢(Sphaeropsis sapinea)的抑制效果显著,抑菌率分别为62.22%和62.78%;经无菌发酵滤液处理后的病原菌菌丝内的丙二醛含量增高,还原糖和可溶性蛋白含量显著降低。WS-FJ9菌株的基因组中含27个不同的次级代谢产物编码基因簇,其中包含编码嗜铁素、细菌素和抗生素等抑菌基因簇;该菌株发酵液在高温、紫外照射和强酸强碱环境条件下及经蛋白酶处理后,其抑菌活性均未受到影响。【结论】多噬伯克霍尔德氏菌WS-FJ9对林木病原菌物具有很好的生防潜力。     

Canopy attachment position influences metabolism and peel constituency of European pear fruit

Background

Inconsistent pear fruit ripening resulting from variable harvest maturity within tree canopies can contribute to postharvest losses through senescence and spoilage that would otherwise be effectively managed using crop protectant and storage regimes. Because those inconsistencies are likely based on metabolic differences, non-targeted metabolic profiling peel of ‘d’Anjou’ pears harvested from the external or internal canopy was used to determine the breadth of difference and link metabolites with canopy position during long-term controlled atmosphere storage.

Results

Differences were widespread, encompassing everything from expected distinctions in flavonol glycoside levels between peel of fruit from external and internal canopy positions to increased aroma volatile production and sucrose hydrolysis with ripening. Some of the most substantial differences were in levels of triterpene and phenolic peel cuticle components among which acyl esters of ursolic acid and fatty acyl esters of p-coumaryl alcohol were higher in the cuticle of fruit from external tree positions, and acyl esters of α-amyrin were elevated in peel of fruit from internal positions. Possibly the most substantial dissimilarities were those that were directly related to fruit quality. Phytosterol conjugates and sesquiterpenes related to elevated superficial scald risk were higher in pears from external positions which were to be potentially rendered unmarketable by superficial scald. Other metabolites associated with fruit aroma and flavor became more prevalent in external fruit peel as ripening progressed and, likewise, with differential soluble solids and ethylene levels, suggesting the final product not only ripens differentially but the final fruit quality following ripening is actually different based on the tree position.

Conclusions

Given the impact tree position appears to have on the most intrinsic aspects of ripening and quality, every supply chain management strategy would likely lead to diverse storage outcomes among fruit from most orchards, especially those with large canopies. Metabolites consistently associated with peel of fruit from a particular canopy position may provide targets for non-destructive pre-storage sorting used to reduce losses contributed by this inconsistency.

     

Isolation and characterization of phytotoxic compounds produced by Phomopsis helianthi

The isolation, chemical characterization and biological activity of two phytotoxic metabolites of Phomopsis helianthi Munt-Cvet et al. is reported. These compounds were identified by spectroscopic methods (UV, IR, 1H and 13C NMR, and MS) as trans-4,6-dihydroxymellein (trans-3-methyl-4,6,8-trihydroxy-3,4-dihyroisocoumarin) and cis-4,6-dihydroxymellein (cis-3-methyl-4,6,8-trihydroxy-3,4-dihydroisocoumarin). This is the first report of the isolation of trans-4,6-dihydroxymellein from fungal cultures and of the production of cis- and trans-4,6-dihydroxymelleins by P. helianthi. Rice was found to be a good substrate for the production of the dihydroxymelleins. Culture extracts of some Italian and French strains of P. helianthi showed different degrees of phytotoxicity towards sunflower leaves and seedlings. The minimum effective doses of trans- and cis-4,6-dihydroxymelleins with different bioassays were 76 and 135 microg per spot (leaf puncture bioassay), 3 and 5 micromol g(-1) fresh tissue (absorption by leaf cutting) and 5 and 2 micromol g(-1) fresh tissue (absorption by cut seedlings), respectively. These compounds may contribute to the severity of the sunflower disease caused by P. helianthi.     

Étude de la pycnogenèse chezPhomopsis mali (Schultz etSacc.)Rob.

Light induces the formation of pycnidia inPhomopsis mali. The induction caused by light can be conserved in darkness. The size and quantity of pycnidia vary with the illuminance. Under low and high illuminances physiologically different pycnidia appear.Phomopsis mali cultivated in darkness in the presence of other microorganisms (fungi or bacteria) can fructify and produces pycnidia similar to those formed under low illuminance and in natural conditions. Our experiments indicate the presence of a pycnogenesis-inducing substance that can exists in different forms and induces the formation of the different pycnidia.     

Diversity and host preference of leaf endophytic fungi in the Iwokrama Forest Reserve, Guyana

Endophytic fungi were isolated from living symptomless leaves of 12 tree species from two locations in the Iwokrama Forest Reserve, Guyana. Sixty-four fungal morphotaxa were characterized from 2492 cultures, which were derived from a total of 2520 sample units. Species of Colletotrichum, Nodulisporium, Pestalotiopsis and Phomopsis were most frequently isolated. Colonization was greater in samples from the midrib than in those from laminar tissue, and slightly greater at the tip of the lamina compared with the base of the leaf. In contrast to studies in temperate ecosystems, no distinct fungal communities were identified for individual plant species, suggesting that the degree of host preference is low. The implications for estimation of fungal diversity in tropical systems are explored.     

Morphological and molecular characterization of Phomopsis vaccinii and additional isolates of Phomopsis from blueberry and cranberry in the eastern United States

Forty isolates of Phomopsis were obtained from twigs and berries of highbush blueberry, Vaccinium corymbosum, and cranberry, Vaccinium macrocarpon, isolated primarily from plants grown in the eastern United States. They were characterized using conidiomatal morphology, conidial dimensions, colony appearance and growth rate, and sequences of ITS rDNA. Based on morphological and molecular similarities, most isolates grouped together with an authentic culture of Phomopsis vaccinii Shear. This taxon is described and illustrated. However, some Phomopsis isolates from Vaccinium differed in colony and conidiomatal morphology from P. vaccinii and, based on ITS sequences, were related to isolates of Phomopsis from diverse hosts. These isolates were excluded from P. vaccinii.     

Cultural Growth Patterns and Incompatibility Reactions in Diaporthe and Phomopsis Populations

Comparative studies were carried out with cultures isolated from Phomopsis condiomata and/or Diaporthe ascomata found on naturally-infected living stems or on overwintered stem fragments of Arctium lappa L., Helianthus annuus L., Glycine max (L.) Merr., and Xanthium italicum Moretti, growing in or nearby the same fields in Vojvodina. Results underscored the heterogeneity of the Phomopsis anamorphs in the D. arctii (Lasch) Trav, complex, Intra-specific vegetative incompatibility groups were distinguished among Phomopsis and/or Diaporthe populations from A. lappa. More than one phytopathogenic Phomopsis and/or Diaporthe species were identified on X. italicum. P. longicolla Hobbs was isolated for the first time in Yugoslavia from soybean stem debris. The holomorph Diaporthe (Phomopsis) helianthi Muntanola- Cvetkovic, Mihaljcevic et Petrov showed distinguishing characteristics with respect to other congeneric fungi.     

Metabolite profiling of Douglas-fir (Pseudotsuga menziesii) field trials reveals strong environmental and weak genetic variation

The primary objective of this study was to assess metabolomics for its capacity to discern biological variation among 10 full-sib families of a Douglas-fir tree breeding population, replicated on two sites. The differential accumulation of small metabolites in developing xylem was examined through metabolite profiles (139 metabolites common to 181 individual trees) generated by gas chromatography mass spectrometry and a series of statistical analyses that incorporated family, site, and tree growth and quantitative phenotypic wood traits (wood density, microfibril angle, wood chemistry and fiber morphology). Multivariate discriminant, canonical discriminant and factor analyses and broad-sense heritabilities revealed that metabolic and phenotypic traits alike were strongly related to site, while similar associations relating to genetic (family) structure were weak in comparison. Canonical correlation analysis subsequently identified correlations between specific phenotypic traits (i.e. tree growth, fibre morphology and wood chemistry) and metabolic traits (i.e. carbohydrate and lignin biosynthetic metabolites), demonstrating a coherent relationship between genetics, metabolism, environmental and phenotypic expression in wood-forming tissue. The association between cambial metabolites and tree phenotype, as revealed by metabolite profiling, demonstrates the value of metabolomics for systems biology approaches to understanding tree growth and secondary cell wall biosynthesis in plants.     

Elm bark beetle boring and feeding deterrents from Phomopsis oblonga

The two groups of Phomopsis oblonga which invade the phloem of stressed elm trees have been investigated for the production in vitro of boring/feeding deterrents for elm bark beetles. The secondary metabolites of this fungus are compared with those of closely-related Phomopsis spp. associated with ash and sycamore. Active compounds isolated from P. oblonga include a novel norsesquiterpene γ-lactone, the tiglic esters of two novel 5,6-dihydro-5-hydroxy-2-pyrones, nectriapyrone, 4-hydroxyphenylethanol, 5-methylmellein, 2-furoic, orsellinic and 3-nitropropanoic acids and mellein-5-carboxylic acid; portensterol and thymine, from P. oblonga, were inactive. (+)-Mellein and furan-2,5-dicarboxylic acid were obtained as minor metabolites of the ash and sycamore Phomopsis strains, which also produced the norsesquiterpene γ-lactone and one of the tiglic esters.     

纯培养下拟茎点霉分生孢子的形成及意义

以18种共23个拟茎点霉菌株为材料,比较了在25℃、12h光照(40w日光灯)/d条件下不同培养基上甲、乙型分生孢子的形成情况。结果表明,苜蓿煎汁+Czapek培养基能促使多数拟茎点霉菌株形成甲、乙两型分生孢子,其形态及大小也与自然寄主上的相一致。根据纯培养获得的形态特征对原始描述缺乙型分生孢子特征记载的Phomopsis durionis, homopsis sterculicola, Phomopsis macadamii, Phomopsis lucumicola和Phomopsis tinea进行了补充描述。