摘要:

為明確感染煙草野火病葉片結(jié)構(gòu)及葉際微生物形態(tài)、碳源代謝能力和群落結(jié)構(gòu)多樣性特征，分別采用掃描電鏡、BIOLOG ECO和Illumina NovaSeq高通量測序技術(shù)研究了感病煙葉與健康煙葉的葉片結(jié)構(gòu)、葉際細(xì)菌和真菌群落結(jié)構(gòu)的差異。結(jié)果表明，感病煙葉表面附著大量細(xì)菌、真菌及菌絲和孢子，煙草葉片受損嚴(yán)重。感病煙葉葉際微生物群落對羧酸類物質(zhì)（丙酮酸甲酯和D-蘋果酸等）的利用能力最強(qiáng)，健康煙葉葉際微生物群落對聚合物（Tween-40和Tween-80等）的利用能力最強(qiáng)，兩組煙葉葉際微生物群落均不能高效利用酚類化合物（2-羥基苯甲酸和4-羥基苯甲酸）。感病和健康煙葉葉際細(xì)菌和真菌在門水平上差異不顯著，但在屬水平上差異顯著。其中，感病煙葉的優(yōu)勢細(xì)菌屬為假單胞菌屬（Pseudomonas，67.43%）和泛菌屬（Pantoea，5.75%），健康煙葉的優(yōu)勢細(xì)菌屬為勞爾氏菌屬（Ralstonia，17.62%）、假單胞菌屬（15.79%）和反芻桿菌屬（Ruminobacter，12.76%）；感病煙葉的優(yōu)勢真菌屬為莖點(diǎn)霉屬（Phoma，24.68%）、鏈格孢屬（Alternaria，22.87%）和Phialocephala（5.72%），健康煙葉的優(yōu)勢真菌屬為枝孢屬（Cladosporium，14.80%）、Sampaiozyma（8.19%）和被孢霉屬（Mortierella，5.23%）。感病煙葉和健康煙葉葉際細(xì)菌和真菌的多樣性、豐富度等均存在顯著差異，暗示著煙草野火病的發(fā)生存在假單胞菌等細(xì)菌與莖點(diǎn)霉等真菌的復(fù)合侵染。

Abstract:

To clarify the characteristics of leaf structure and phyllosphere microbial morphology, carbon source metabolic capacity, and community structure diversity for tobacco leaves affected by wildfire disease, scanning electron microscopy, BIOLOG ECO, and Illumina NovaSeq high-throughput sequencing technologies were used to investigate the differences in leaf structure, phyllosphere bacteria and fungal communities between infected and healthy tobacco leaves. The results showed that a significant number of bacterial hyphae, fungal hyphae, and spores adhered to the surfaces of infected tobacco leaves that were severely damaged. The phyllosphere microbial communities of the infected tobacco leaves had the highest utilization capacity for carboxylic acids (methyl pyruvate and D-malate, etc.), whereas those of healthy tobacco leaves had the highest utilization capacity for polymers (Tween-40 and Tween-80, etc.). Both leaf groups were inefficient in the utilization of phenolic compounds (2-hydroxybenzoic acid and 4-hydroxybenzoic acid). Although no significant differences were observed in the phyllosphere bacterial and fungal communities between the infected and healthy tobacco leaves at phylum level, significant differences were observed at the genus level. Dominant bacterial genera of the infected tobacco leaves were Pseudomonas (67.43%) and Pantoea (5.75%), whereas Ralstonia (17.62%), Pseudomonas (15.79%) and Ruminobacter (12.76%) dominated in the healthy samples. Dominant fungal genera were Phoma (24.68%), Alternaria (22.87%) and Phialocephala (5.72%) on the infected tobacco leaves, whereas Cladosporium (14.80%), Sampaiozyma (8.19%) and Mortierella (5.23%) on the healthy tobacco leaves. Significant differences in diversity and richness between phyllosphere bacterial and fungal communities of the infected and healthy tobacco leaves indicated that a composite infection of bacteria such as Pseudomonas spp. and fungi such as Phoma spp. contributed to the progression of tobacco wildfire disease.

圖  1   不同癥狀煙葉的形態(tài)掃描電鏡觀察與特征比較

A和B為感病煙葉正面危害癥狀，C和D為感病煙葉背面危害癥狀；a為健康煙葉表面電鏡組織形態(tài)，b、c、d均為感病煙葉表面組織形態(tài)。

Fig.  1   Morphological observation by scanning electron microscopy and characterization of tobacco leaves with different symptoms

圖  2   健康煙葉與感病煙葉葉際微生物的碳源代謝圖譜

圖例顏色表示Biolog OmniLog R系統(tǒng)AWCD值的大小。

Fig.  2   Carbon source metabolism map of phyllosphere microorganisms of infected and healthy tobacco leaves

圖  3   健康（LPSJ）和感?。↙PSB）煙葉樣品細(xì)菌（A）和真菌（B）ASV分布Venn圖

Fig.  3   Venn diagrams of ASV distribution for bacteria (A) and fungi (B) in healthy (LPSJ) and infected (LPSB) tobacco leaf samples

圖  4   健康與感病煙葉葉際細(xì)菌（A）、真菌（B）物種進(jìn)化樹與細(xì)菌門（C）、屬（E）和真菌門（D）、屬（F）水平組間的相對豐度

A、B左側(cè)圖例為樣本信息，右側(cè)圖例為屬水平物種對應(yīng)的門水平的分類信息；圖C、D、E、F圖例中Others表示圖中10個(gè)門/屬之外的其他所有門/屬的相對豐度之和。

Fig.  4   Evolutionary trees of phyllosphere microorganisms, bacteria (A) and fungi (B), on healthy and infected tobacco leaves, and relative abundances of bacterial phylum (C), bacterial genus (E), fungal phylum (D) and fungal genus (F) groups

圖  5   健康（LPSJ）和感?。↙PSB）煙葉樣本中細(xì)菌（A）與真菌（B）群落的主成分分析（PCA）

橫坐標(biāo)表示第一主成分，縱坐標(biāo)表示第二主成分，百分比表示主成分對樣本差異的貢獻(xiàn)值。

Fig.  5   Principal component analysis (PCA) of bacterial (A) and fungal (B) communities of healthy (LPSJ) and infected (LPSB) tobacco leaf samples

圖  6   細(xì)菌與真菌群落組間進(jìn)化分支圖與LDA值分布柱形圖

A.細(xì)菌進(jìn)化分支圖；B.細(xì)菌LDA分布圖；C.真菌進(jìn)化分支圖；D.真菌LDA分布圖。在進(jìn)化分支圖中，由內(nèi)至外輻射的圓圈代表了由門至屬（或種）的分類級別。在不同分類級別上的每一個(gè)小圓圈代表該水平下的一個(gè)分類，小圓圈直徑大小與相對豐度大小呈正比。LDA值分布柱狀圖中展示了LDA score大于設(shè)定值（默認(rèn)設(shè)置為4）的物種，即組間具有統(tǒng)計(jì)學(xué)差異的Biomarker。

Fig.  6   Intergroup evolutionary branching diagrams of bacterial and fungal communities and bar charts of LDA value distribution

表  1   不同煙葉樣品的序列數(shù)據(jù)

Tab.  1   Sequence data of different tobacco leaf samples

樣品 有效序列/條 堿基數(shù)/nt 平均長度/nt 細(xì)菌 真菌 細(xì)菌 真菌 細(xì)菌 真菌 LPSB1 76 812 55 540 31 194 278 14 315 321 406 258 LPSB2 82 984 65 834 33 823 340 15 949 946 408 242 LPSB3 60 323 72 532 25 132 199 15 306 587 417 211 LPSB4 75 970 64 386 31 145 999 13 647 568 410 212 LPSJ1 70 754 64 143 28 782 147 17 015 930 407 265 LPSJ2 83 541 70 913 33 943 411 17 923 390 406 253 LPSJ3 68 900 68 345 28 034 166 17 345 137 407 254 LPSJ4 85 154 69 099 34 582 234 15 719 312 406 227

表  2   樣品組間α多樣性指數(shù)①

Tab.  2   Alpha diversity index between sample groups

樣品 Shannon指數(shù) Simpson指數(shù) Chao1指數(shù) Pielou_e指數(shù) 覆蓋度 細(xì)菌 LPSJ 4.28±0.90a 0.89±0.06a 58.89±31.49a 0.77±0.43a 0.982 LPSB 2.95±1.19a 0.70±0.18a 71.69±39.84a 0.55±0.22a 0.963 真菌 LPSJ 6.10±1.45b 0.94±0.06a 480.99±180.07b 0.69±0.12b 1.000 LPSB 2.78±1.77a 0.61±0.30a 159.03±99.83a 0.38±0.20a 1.000注：①表中數(shù)據(jù)為平均值±標(biāo)準(zhǔn)差，同列不同字母表示有顯著性差異（P＜0.05）。 [1]

Xin X F, Kvitko B, He S Y. Pseudomonas syringae: What it takes to be a pathogen[J]. Nature Reviews Microbiology, 2018, 16(5): 316-328.

[2] 陳瑞朋, 盧凱, 張敏, 等. 煙草野火病菌特異性檢測引物篩選及應(yīng)用[J]. 中國煙草科學(xué), 2018, 39(1): 72-76.

CHEN Ruipeng, LU Kai, ZHANG Min, et al. Screening and application of Pseudomonas syringae pv. Tabaci-specific primers[J]. Chinese Tobacco Science, 2018, 39(1): 72-76.

[3]

Kusstatscher P, Cernava T, Harms K, et al. Disease incidence in sugar beet fields is correlated with microbial diversity and distinct biological markers[J]. Phytobiomes Journal, 2019, 3(1): 22-30. doi: 10.1094/PBIOMES-01-19-0008-R

[4] 劉暢, 汪漢成, 謝紅煉, 等. 感染赤星病煙草葉際細(xì)菌的多樣性分析[J]. 煙草科技, 2020, 53(2): 8-14. doi: 10.16135/j.issn1002-0861.2019.0114

LIU Chang, WANG Hancheng, XIE Honglian, et al. Biodiversity analysis of phyllosphere bacterial genus from tobacco leaves infected by brown spot disease[J]. Tobacco Science & Technology, 2020, 53(2): 8-14. doi: 10.16135/j.issn1002-0861.2019.0114

[5] 黃宇, 汪漢成, 陳乾麗, 等. 感染白粉病煙株典型病級葉際真菌群落結(jié)構(gòu)與多樣性分析[J]. 煙草科技, 2021, 54(4): 8-14. doi: 10.16135/j.issn1002-0861.2019.0475

HUANG Yu, WANG Hancheng, CHEN Qianli, et al. Community structure and diversity of phyllosphere fungi in tobacco plants with typically infected tobacco powdery mildew[J]. Tobacco Science & Technology, 2021, 54(4): 8-14. doi: 10.16135/j.issn1002-0861.2019.0475

[6] 孫美麗, 史彩華, 肖本青, 等. 煙草靶斑病葉際微生物群落結(jié)構(gòu)與多樣性分析[J]. 煙草科技, 2023, 56(4): 1-9. doi: 10.16135/j.issn1002-0861.2022.0891

SUN Meili, SHI Caihua, XIAO Benqing, et al. Composition and diversity of phyllospheric microbial community in tobacco leaves infected by tobacco target spot disease[J]. Tobacco Science & Technology, 2023, 56(4): 1-9. doi: 10.16135/j.issn1002-0861.2022.0891

[7] 劉亭亭, 汪漢成, 孫美麗, 等. 波爾多液對煙草葉際微生物群落結(jié)構(gòu)與代謝功能的影響[J]. 農(nóng)藥學(xué)學(xué)報(bào), 2022, 24(6): 1446-1455.

LIU Tingting, WANG Hancheng, SUN Meili, et al. Effects of Bordeaux mixture on the community structure and metabolic function of tobacco phyllosphere microorganisms[J]. Chinese Journal of Pesticide Science, 2022, 24(6): 1446-1455.

[8] 向立剛, 汪漢成, 羅飛, 等. 感染青枯病與黑脛病煙株的根際土壤、根及莖稈微生物代謝特征分析[J]. 煙草科技, 2023, 56(3): 17-24. doi: 10.16135/j.issn1002-0861.2022.0394

XIANG Ligang, WANG Hancheng, LUO Fei, et al. Metabolic characteristics of microorganisms in rhizosphere soil, roots, and stalks of tobacco plants infected with bacterial wilt and black shank[J]. Tobacco Science & Technology, 2023, 56(3): 17-24. doi: 10.16135/j.issn1002-0861.2022.0394

[9] 俞艷玲, 邢旺興, 陳士景. 7種紅曲霉的掃描電鏡觀察[J]. 浙江中醫(yī)學(xué)院學(xué)報(bào), 2005(1): 78-79.

YU Yanling, XING Wangxing, CHEN Shijing. Study on the physiological characteristics of seven monascus fungi by scanning electron microscope[J]. Journal of Zhejiang College of Traditional Chinese Medicine, 2005(1): 78-79.

[10]

Dai Y F, Wu X M, Wang H C, et al. Spatio-temporal variation in the phyllospheric microbial biodiversity of Alternaria alternata-infected tobacco foliage[J]. Frontiers in Microbiology, 2022, 13: 920109.

[11] 吳盼云, 晁群芳, 趙亞光, 等. 克拉瑪依石油污染土壤微生物群落結(jié)構(gòu)及其代謝特征[J]. 基因組學(xué)與應(yīng)用生物學(xué), 2019, 38(5): 2062-2069.

WU Panyun, CHAO Qunfang, ZHAO Yaguang, et al. Microbial community structure and metabolic characteristics of oil-contaminated soil in Karamay[J]. Genomics and Applied Biology, 2019, 38(5): 2062-2069.

[12]

Zhang L, Wang Y, Xia X Y, et al. Altered gut microbiota in a mouse model of Alzheimers disease[J]. Journal of Alzheimer's Disease, 2017, 60(4): 1241-1257.

[13]

Lu L H, Yin S X, Liu X, et al. Fungal networks in yield-invigorating and -debilitating soils induced by prolonged potato monoculture[J]. Soil Biology and Biochemistry, 2013, 65: 186-194.

[14]

Mago? T, Salzberg S L. FLASH: Fast length adjustment of short reads to improve genome assemblies[J]. Bioinformatics, 2011, 27(21): 2957-2963.

[15]

Edgar R C, Haas B J, Clemente J C, et al. UCHIME improves sensitivity and speed of chimera detection[J]. Bioinformatics, 2011, 27(16): 2194-2200.

[16]

Li M J, Shao D T, Zhou J C, et al. Signatures within esophageal microbiota with progression of esophageal squamous cell carcinoma[J]. Chinese Journal of Cancer Research, 2020, 32(6): 755-767.

[17] 陳燾, 周瑋, 李宏光, 等. 煙草野火病的發(fā)生及綜合防治研究進(jìn)展[J]. 基因組學(xué)與應(yīng)用生物學(xué), 2018, 37(1): 469-476.

CHEN Tao, ZHOU Wei, LI Hongguang, et al. Research progress in disease occurrence and integrated control of tobacco wildfire[J]. Genomics and Applied Biology, 2018, 37(1): 469-476.

[18]

Lee N M, Lee B H. Thermodynamics on the micellization of pure cationic(DTAB, TTAB, CTAB), nonionic(Tween-20, Tween-40, Tween-80), and their mixed surfactant systems[J]. Journal of the Korean Applied Science and Technology, 2013, 30(4): 679-687.

[19]

Suksiri P, Ismail A, Sirirattanachatchawan C, et al. Enhancement of large ring cyclodextrin production using pretreated starch by glycogen debranching enzyme from Corynebacterium glutamicum[J]. International Journal of Biological Macromolecules, 2021, 193(PA): 81-87.

[20]

Wu N, Wu X Y, Zhang M Y, et al. Metabolic engineering of Aspergillus niger for accelerated malic acid biosynthesis by improving NADPH availability[J]. Biotechnology Journal, 2024, 19(5): e2400014.

[21]

Groza N V, Yarkova T A, Gessler N N, et al. Synthesis of benzoic acid esters and their antimicrobial activity[J]. Pharmaceutical Chemistry Journal, 2024, 58(4): 625-630.

[22] 吳小軍, 汪漢成, 孫美麗, 等. 煙草角斑病葉際微生物群落結(jié)構(gòu)與多樣性分析[J]. 煙草科技, 2023, 56(10): 1-10. doi: 10.16135/j.issn1002-0861.2023.0282

WU Xiaojun, WANG Hancheng, SUN Meili, et al. Composition and diversity of phyllosphere microbial community on tobacco leaves infected with angular leaf spot[J]. Tobacco Science & Technology, 2023, 56(10): 1-10. doi: 10.16135/j.issn1002-0861.2023.0282

[23]

Si H Y, Cui B, Liu F, et al. Microbial community and chemical composition of cigar tobacco (Nicotiana tabacum L. ) leaves altered by tobacco wildfire disease[J]. Plant Direct, 2023, 7(12): e551.

[24] 劉宇星, 董醇波, 邵秋雨, 等. 葉際微生物與植物健康研究進(jìn)展[J]. 微生物學(xué)雜志, 2022, 42(2): 88-98.

LIU Yuxing, DONG Chunbo, SHAO Qiuyu, et al. Advances on phyllosphere microorganisms and their association with plant health[J]. Journal of Microbiology, 2022, 42(2): 88-98.

[25] 江艷, 桑維鈞, 曾爾玲, 等. 煙草莖點(diǎn)病在貴州省的發(fā)生及病原鑒定[J]. 江蘇農(nóng)業(yè)科學(xué), 2018, 46(10): 92-95.

JIANG Yan, SANG Weijun, ZENG Erling, et al. Identification of pathogenic fungus causing tobacco black spot stalk in Guizhou Province[J]. Jiangsu Agricultural Sciences, 2018, 46(10): 92-95.

[26]

Wei L L, Chen B, Li J W, et al. Resistance mechanism of Phomopsis longicolla to fludioxonil is associated with modifications in PlOS1, PlOS4 and PlOS5[J]. Pesticide Biochemistry and Physiology, 2024, 201: 105862.

[27]

Eduardo L A O D, Antonio P L, Antonio J L, et al. Addressing coffee crop diseases: forecasting Phoma leaf spot with machine learning[J]. Theoretical and Applied Climatology, 2023, 155(3): 2261-2282.

[28]

Hansen B C, Gilley M A, Berghuis B G, et al. Effect of fungicide and timing of application on management of Phoma black stem of cultivated sunflowers in the United States[J]. Plant Disease, 2024, 108(7): 2017-2026.

[29] 李紀(jì)潮, 張金渝, 蔡明姬, 等. 草果葉斑病類致病菌對新型低毒藥劑的敏感性及交互抗性[J]. 南方農(nóng)業(yè)學(xué)報(bào), 2022, 53(8): 2153-2160.

LI Jichao, ZHANG Jinyu, CAI Mingji, et al. Sensitivity and cross-resistance of pathogen of Amomum tsaoko leaf spot to new low-toxic fungicides[J]. Journal of Southern Agriculture, 2022, 53(8): 2153-2160.

相關(guān)知識

煙草根腐病發(fā)病與健康根際微生物生物群落分析及球毛殼菌NP2的生防效果研究
炭疽病發(fā)病草莓與健康草莓根際細(xì)菌群落結(jié)構(gòu)及功能差異
煙草微生物組研究取得新進(jìn)展
科研丨復(fù)旦: 季節(jié)性變化驅(qū)動(dòng)水生根際微生物群落結(jié)構(gòu)和功能特性的變化(國人佳作)
《自然》：“葉圈”健康有多重要？中美科學(xué)家聯(lián)合發(fā)表植物葉際微生物群領(lǐng)域的新進(jìn)展
茶樹葉面真菌與內(nèi)生真菌群落結(jié)構(gòu)的比較
微生物多樣性與人類健康.docx
熱帶植物的結(jié)構(gòu)與生態(tài).pptx
健康廣藿香與患病廣藿香根際土壤微生物群落的結(jié)構(gòu)
藥用大黃黑粉病發(fā)病部位及根際土壤微生物群落多樣性研究

網(wǎng)址: 煙草野火病葉際微生物群落結(jié)構(gòu)多樣性與碳源代謝表征 http://www.gysdgmq.cn/newsview1904799.html