Endophytic diversity in citrus varieties in different provinces
The native bacterial endophytic communities of healthy, symptomatic, and asymptomatic citrus leaves in nine citrus growing regions was assessed in 2016–2018, and Fig. 2 show the endophytic microbial diversity of different citrus varieties from different citrus growing regions in China. The endophyte communities varied between specific varieties from different sites and between different varieties from the same site. Diverse endophytic bacteria were recovered from the same and different varieties located in multiple site or individual sites, respectively. Fujian province had a large range of citrus varieties, which resulted in the maximum endophyte isolation frequency, with C. reticulata Blanco having more endophytes in Fujian. However, the number of endophytes depends on the number of citrus varieties in each specific location; for example, the high endophyte isolation frequency in Fujian province was due to the collection of samples from 11 citrus varieties. Fewer endophytes were recovered from C. grandis (L.) Osbeck cv. Guanximiyou in Fujian province. Chongqing province had only 30 endophytes. Among all of the citrus varieties from different provinces, Valencia Orange had maximum endophyte species (22). In addition, citrus varieties in different and same provinces were different in terms of endophyte recovery. The total number of endophytes isolated from most of the citrus varieties was 104–106 CFU/g of leaves, with no significant differences between citrus varieties (Table S1).
Total endophytic bacteria from 24 citrus varieties from nine citrus growing regions in China. Provinces with different citrus varieties have diverse endophytes. The bar representing only variety 8 corresponds to the Zhejiang province. The dark grey bars represent the non-repeated varieties in different provinces. Citrus varieties are labeled with the same numbers in different provinces: 1, Citrus reticulata Blanco; 2, C. sinensis (L.) Osbeck; 3, C. reticulata cv. Tankan; 4, C. unshiu Marcov. forma Miyagawa-wase × C. sinensis (L.) Osbeck; 5, C. reticulata cv. Shatangju; 6, C. maxima cv. Sanhongmiyou; 7, C. reticulata Blanco v. Gonggan; 8, C. reticulata; 9, C. reticulata cv. Suavissima; 10, C. grandis (L.) Osbeck cv. Guanximiyou; 11, C. sinensis; 12, C. tangerine; 13, C. unshiu Marc; 14, Huangyan; 15, Juhong orange; 16, C. reticulate (L.) Blanco cv. Nanfengmiju; 17, Fortunella margarita (L.) Swingle; 18, Valencia orange; 19, C. limon (L.) Burm. f.; 20, C. reticulata cv. Ponkan; 21, C. sinensis Osb. (navel orange); 22, Puzao; 23, Tezao; 24, Chishu.
Dominant endophytes in citrus leaves
The dominant bacterial endophytes isolated from the various citrus varieties were Bacillus subtilis, Bacillus sp., B. velezensis, B. amyloliquefaciens, B. megaterium, B. tequilensis, Curtobacterium luteum, Gammaproteobacterium symbiont of Plautia stali, Microbacterium testaceum, B. licheniformis, B. methylotrophicus, B. pumilus, B. vallismortis, Curtobacterium citreum, C. herbarum, C. luteum, C. oceanosedimentum, Curtobacterium sp., Geobacillus stearothermophilus, Staphylococcus epidermidis, B. aryabhattai, B. huizhouensis, B. hunanensis, B. koreensis, B. niacini, B. pseudomycoides, B. stratosphericus, Brachybacterium sp., C. oceanosedimentum, Enhydrobacter sp., Enterobacter sp., Lentibacillus populi, Lysinibacillus massiliensis, Massilia sp., Moraxella osloensis, Oceanobacillus kimchii, Paenibacillus amylolyticus, P. silvae, Pantoea eucrina, P. septica, Proteus mirabilis, Sphingobium yanoikuyae, S. endophytica, S. paucimobilis, S. yunnanensis, S. saprophyticus, Staphylococcus sp., and Terribacillus sp. (Table 1). Figure S4 shows how pure cultures of dominant endophytes were obtained by streaking individual colonies on LB agar. Phylogenetic analyses of the dominant isolates are shown in Fig. 3.
Phylogenetic tree of dominant endophytes based on the 16S rRNA gene. The evolutionary history was inferred using the Maximum Likelihood method based on the Kimura two-parameter model and the analysis involved 11 nucleotide sequences. The percentage of trees in which the associated taxa clustered together is shown next to the branches. All positions with gaps and missing data were eliminated. The analysis was conducted in MEGA7.
Detection of CLas
All collected citrus leaves were investigated for the presence of CLas with CLas-specific primers37. The number of CLas copies was different between symptomatic and asymptomatic plants, with symptomatic plants having lower cycle threshold (CT) values and more CLas copies. No CLas was observed in healthy (uninfected) plants. After visually assessing the leaves as symptomatic or healthy/asymptomatic, the healthy/asymptomatic leaves were distinguished based on CT values and the number of CLas copies, with CT values >32, 25–32, and <25 (100–101, 104, and 106CLas copies/g leaves) representing healthy, asymptomatic, and symptomatic states, respectively (Table 2).
Comparison of endophytes based on disease state
The endophyte isolation frequency was significantly different between leaves with different disease states, with the highest numbers in the healthy leaves and the lowest in the symptomatic leaves (p < 0.05) (Fig. 4). Among the 114 bacterial endophytes isolated from the healthy leaves, the most dominant were B. subtilis, B. velezensis, C. luteum, S. endophytica, B. tequilensis, P. amylolyticus, and M. testaceum. Among the 41 endophytes isolated from the symptomatic trees, Bacillus sp. was the most dominant endophyte followed by Curtobacterium, and among the 58 endophytes isolated from the asymptomatic leaves, Bacillus sp. and B. megaterium were the most dominant endophytes. None of the other endophytes in healthy leaves were found in the symptomatic or asymptomatic leaves. Although Bacillus sp. was frequently isolated from all leaves, the isolation frequencies were considerably lower in the asymptomatic and symptomatic leaves compared to the healthy leaves. Most of the endophyte species were isolated at low frequencies from the various citrus varieties and regions.
Total isolation frequency of native endophytic bacteria in healthy, symptomatic, and asymptomatic citrus trees from different provinces in China. (a) Total number of endophytes by huanglongbing disease state (confirmed using qPCR targeting the ribosomal protein L12 [rplL] of the Candidatus Liberibacter asiaticus [CLas] pathogen). (b) The disease states of the leaves were assessed before endophyte isolation from specific citrus varieties. Data were analyzed using analysis of variance (ANOVA) followed by Duncan’s multiple range test (p < 0.05). (a–c) Indicate significant differences among leaves with different disease states and error bars indicate the standard error of the mean (SEM).
Number of endophyte species among different citrus varieties
We assessed the native endophyte communities in 24 citrus varieties. Valencia orange had the maximum number of endophyte species (22 species), while C. reticulata cv. Ponkan had the second highest number of endophyte species (14 species), followed by C. reticulata Blanco, C. unshiu Marcov. forma Miyagawa-wase × C. sinensis (L.) Osbeck, and C. reticulata (which all had 12 species) (Table 3).
Endophyte species common to multiple citrus varieties
There were 19 endophyte species in many of the citrus varieties from each region. These endophytes were B. subtilis, Bacillus sp., B. velezensis, B. amyloliquefaciens, B. megaterium, B. tequilensis, C. luteum, Gammaproteobacterium symbiont of Plautia stali, M. testaceum, B. licheniformis, B. methylotrophicus, B. pumilus, B. vallismortis, C. citreum, C. herbarum, C. oceanosedimentum, Curtobacterium sp., G. stearothermophilus, and S. epidermidis. They were isolated from many of the citrus varieties, including C. reticulata Blanco, C. sinensis (L.) Osbeck, C. reticulata cv. Tankan, C. unshiu Marcov. forma Miyagawa-wase × C. sinensis (L.) Osbeck, C. reticulata cv. Shatangju, C. maxima cv. Sanhongmiyou, C. reticulata Blanco var. Gonggan, C. reticulata, C. reticulata cv. Suavissima, C. grandis (L.) Osbeck cv. Guanximiyou, C. sinensis, C. tangerine, C. unshiu Marc, Huangyan, Juhong orange, C. reticulata (L.) Blanco cv. Nanfengmiju, Fortunella margarita (L.) Swingle, Valencia orange, C. limon (L.) Burm. f., C. reticulata cv. Ponkan, and C. sinensis Osb. (navel orange).
Endophyte species in leaves with different disease states
The dominant endophytes in different citrus varieties were compared, and B. subtilis was found to be the most frequent species in the healthy citrus plants and it was also recovered from a few asymptomatic and symptomatic trees (Fig. 5). B. subtilis showed significant differences between healthy trees and both symptomatic and asymptomatic trees (p < 0.05). The other dominant species across all disease states were Bacillus sp., B. velezensis, B. amyloliquefaciens, B. megaterium, B. tequilensis, C. luteum, Gammaproteobacterium symbiont of Plautia stali, and M. testaceum. In contrast, C. luteum, S. endophytica, P. amylolyticus, M. osloensis, and P. septica were frequently isolated only from healthy leaves. Interestingly, only Bacillus sp. was frequently recovered from asymptomatic leaves (p < 0.05), and it may provide resistance against CLas.
Comparison of endophyte isolation frequency by huanglongbing disease state. Means with the same letter are not significantly different based on the Tukey test (p < 0.05) and error bars indicate the standard error of the mean (SEM).
Source: Ecology - nature.com
