The influence of genetics,defensive chemistry and the fungal microbiome on disease outcome in whitebark pine trees

The invasive fungal pathogen Cronartium ribicola infects and kills whitebark pine (Pinus albicaulis) throughout western North America. Whitebark pine has been proposed for listing under the Endangered Species Act in the USA, and the loss of this species is predicted to have severe impacts on ecosystem composition and function in high‐elevation forests. Numerous fungal endophytes live inside whitebark pine tissues and may influence the severity of C. ribicola infection, either directly by inhibition of pathogen growth or indirectly by the induction of chemical defensive pathways in the tree. Terpenes, a form of chemical defence in pine trees, can also influence disease. In this study, we characterized fungal endophyte communities in whitebark pine seedlings before and after experimental inoculation with C. ribicola, monitored disease progression and compared fungal community composition in susceptible vs. resistant seedlings in a common garden. We analysed the terpene composition of these same seedlings. Seed family identity or maternal genetics influenced both terpenes and endophyte communities. Terpene and endophyte composition correlated with disease severity, and terpene concentrations differed in resistant vs. susceptible seedlings. These results suggest that the resistance to C. ribicola observed in natural whitebark pine populations is caused by the combined effects of genetics, endophytes and terpenes within needle tissue, in which initial interactions between microbes and hosts take place. Tree genotype, terpene and microbiome combinations associated with healthy trees could help to predict or reduce disease severity and improve outcomes of future tree breeding programmes.     

Interactions of Neotyphodium gansuense, Achnatherum inebrians, and plant-pathogenic fungi

Interactions of Neotyphodium gansuense, Achnatherum inebrians, and nine fungal pathogens were studied by tests of inhibition of four fungal pathogens by Neotyphodium endophytes in vitro and by inoculation of nine fungal pathogens on detached leaves of endophyte-infected (E+) and endophyte-free (E−) plants. Compared with the controls, most isolates of N. gansuense significantly inhibited the growth in vitro of, in decreasing order of inhibition, Bipolaris sorokiniana, Curvularia lunata, Fusarium acuminatum, and Alternaria alternata. Inhibition zones appeared between pathogens and some isolates of N. gansuense. Some isolates of N. gansuense significantly inhibited sporulation of B. sorokiniana, A. alternata, and C. lunata. However, there was no significant inhibition of F. acuminatum and a few isolates significantly increased sporulation. The leaf inoculation trial indicated that almost all fungal pathogens were able to cause lesions on detached leaves regardless of endophyte status. Both the number and size of disease lesions on E+ A. inebrians leaves caused by A. alternata, F. chlamydosporum, F. oxysporum, and F. solani were reduced compared with those on E− leaves. Only lesion numbers (not size) of Ascochyta leptospora leaf spots were significantly reduced on E+ leaves compared with E− leaves. Conversely, only the length of Ascochyta leptospora leaf spots were significantly smaller on E+ leaves than on E− leaves; numbers of lesions were not significantly affected. C. lunata was strongly pathogenic to both E+ and E− leaves and numerous lesions developed and merged into patches, the leaf surface was covered and the leaf rotted away.     

Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms

DNA sequencing continues to decrease in cost with the Illumina HiSeq2000 generating up to 600 Gb of paired-end 100 base reads in a ten-day run. Here we present a protocol for community amplicon sequencing on the HiSeq2000 and MiSeq Illumina platforms, and apply that protocol to sequence 24 microbial communities from host-associated and free-living environments. A critical question as more sequencing platforms become available is whether biological conclusions derived on one platform are consistent with what would be derived on a different platform. We show that the protocol developed for these instruments successfully recaptures known biological results, and additionally that biological conclusions are consistent across sequencing platforms (the HiSeq2000 versus the MiSeq) and across the sequenced regions of amplicons.     

A biologist's guide to de novo genome assembly using next-generation sequence data: A test with fungal genomes

We offer a guide to de novo genome assembly¹ using sequence data generated by the Illumina platform for biologists working with fungi or other organisms whose genomes are less than 100 Mb in size. The guide requires no familiarity with sequencing assembly technology or associated computer programs. It defines commonly used terms in genome sequencing and assembly; provides examples of assembling short-read genome sequence data for four strains of the fungus Grosmannia clavigera using four assembly programs; gives examples of protocols and software; and presents a commented flowchart that extends from DNA preparation for submission to a sequencing center, through to processing and assembly of the raw sequence reads using freely available operating systems and software.     

The co-occurrence of ectomycorrhizal,arbuscular mycorrhizal,and dark septate fungi in seedlings of four members of the Pinaceae

Although roots of species in the Pinaceae are usually colonized by ectomycorrhizal (EM) fungi, there are increasing reports of the presence of arbuscular mycorrhizal (AM) and dark septate endophytic (DSE) fungi in these species. The objective of this study was to determine the colonization patterns in seedlings of three Pinus (pine) species (Pinus banksiana, Pinus strobus, Pinus contorta) and Picea glauca x Picea engelmannii (hybrid spruce) grown in soil collected from a disturbed forest site. Seedlings of all three pine species and hybrid spruce became colonized by EM, AM, and DSE fungi. The dominant EM morphotype belonged to the E-strain category; limited colonization by a Tuber sp. was found on roots of Pinus strobus and an unknown morphotype (cf. Suillus–Rhizopogon group) with thick, cottony white mycelium was present on short roots of all species. The three fungal categories tended to occupy different niches in a single root system. No correlation was found between the percent root colonized by EM and percent colonization by either AM or DSE, although there was a positive correlation between percent root length colonized by AM and DSE. Hyphae and vesicles were the only AM intracellular structures found in roots of all species; arbuscules were not observed in any roots.     

Endophytic fungi associated with the Antarctic grass <Emphasis Type="Italic">Deschampsia antarctica</Emphasis> Desv. (<Emphasis Type="Italic">Poaceae</Emphasis>)

Deschampsia antarctica Desv. (Poaceae) represents one of the two vascular plants that have colonized the Antarctic continent, which is usually exposed to extreme environmental conditions. In this work, we have characterized the endophytic fungi associated with the leaves of D. antarctica. Endophytic fungi were recovered from 91 individual plants from different points of Admiralty Bay at King George Island, Antarctica. A total of 26 fungal isolates were obtained from 273 leaf fragments. All isolates were identified by analysis of the sequences of the internal transcribed spacer region (ITS) of the rDNA. Alternaria and Phaeosphaeria were the most frequent genera associated with the plant. Other fungal isolates were identified as Entrophospora sp. and several undescribed Ascomycete species. An interesting result was obtained for the isolates UFMGCB 215 and UFMGCB 262, which were related to fungi associated with bryophytes present in boreal ecosystems. Some isolates showed low identity in the ITS sequences to sequences of fungal species deposited in GenBank, suggesting that these fungi could be new species. This work is the first report on fungal endophytes associated with leaves of the Antarctic grass D. antarctica.     

真菌菌剂改善烟叶品质的初步研究*

利用分离自不同生境烟叶上的7个真菌菌株菌剂处理上部叶烤烟烟丝,分析表明,处理后烟叶内与品质相关的主要化学成分含量发生了改变,烟叶品质得到改善。其中菌株BF03、BF06及BF63使烟叶内糖、氮、烟碱、蛋白质等主要化学成分含量及各种成分之间比例趋于平衡。评吸结果表明,经供试菌剂处理的烟叶香气质提高,香气量增加,刺激性减小,余味舒适,烟叶品质明显高于对照。     

高通量测序技术在植物内生菌领域研究中的应用

植物内生菌是一类能够在植物体内度过部分生命周期而不会引起宿主病害的微生物,是种类和功能十分丰富的微生物资源。随着测序技术的进步,在微生物领域中关于植物内生菌的研究愈发深入。Sanger测序的定向验证性以及较低的测序成本使得该测序技术沿用至今,但其测序的通量较低,不适用于更深层次的内生菌基因组研究。本文简要概述了植物内生菌的研究历程,并综述了以高通量为技术特点的下一代测序(next-generation sequencing,NGS)和以单分子实时测序技术(single-molecule real-time,SMRT)为代表的第三代测序技术在植物内生菌研究领域的应用,总结了不同测序技术在实际研究中的优势和局限性,并探讨了如何根据研究需求选择合适的测序技术,希望为研究者进一步发掘植物内生菌的潜在价值提供参考。     

Diversity and antimicrobial activity of endophytic fungi isolated from Nyctanthes arbor-tristis, a well-known medicinal plant of India

Endophytic fungi from Nyctanthes arbor-tristis were isolated and evaluated for their antimicrobial activity. A total of 19 endophytic fungi were isolated from 400 segments of healthy leaf and stem tissues of N. arbor-tristis. Eighteen endophytic fungi were obtained from leaf, while only ten from stem. Alternaria alternata had the highest colonization frequency (15.0%) in leaf, whereas Cladosporium cladosporioides ranked first in stem with a colonization frequency of 12%. The diversity and species richness were found higher in leaf tissues than in stem. The similarity indices between leaf and stem were 0.473 for Jaccard’s and 0.642 for the Sorenson index, respectively. Of 16, 12 (75%) endophytic fungal extracts showed antibacterial activity against either one or more pathogenic bacteria. The endophytic Nigrospora oryzae showed maximum inhibition against Shigella sp. and Pseudomonas aeruginosa. The leaf endophytes Colletotrichum dematium and Chaetomium globosum exhibited a broad range of anibacterial activity and were active against Shigella flexnii, Shigella boydii, Salmonella enteritidis, Salmonella paratyphi, and P. aeruginosa. Nine out of 16 (56.25%) endophytic fungi exhibited antifungal activity to one or more fungal pathogens. Colletotrichum dematium inhibited 55.87% of the radial growth of the phytopathogen Curvularia lunata. The antimicrobial activity of these endophytic microorganisms could be exploited in the biotechnological, medicinal, and agricultural industries.