Poplar bacterial canker disease was first discovered in France in 1960, and gradually spread throughout Europe, Canada and the United States. Since Xiang et al. (2001) discovered poplar bacterial canker caused by Erwinia in Zhaodong in 1982, the condition and etiology of this disease have been systematically studied. It was preliminarily determined that poplar bacterial canker in Northeast China was caused by ice nucleation bacteria (ING) in Gracilicutes (Xiang et al., 2001).  Related studies have shown that ING include: Pseudomonas. ananas Scrrano, Erwinia. Urodovora Dye, Pseudomonas. syringae pv. syringae Van Hall, Pseudomonas.syringae pv. Lachrymans (Smith & Bryan) Dye), Pseudomonas syringae pvs. and Erwinia herbicola (Lohins) Dye (Ren, 2011). There are various hosts of poplar bacterial canker pathogen, without obvious host and regional specificity (Zhao, 1992). Relevant studies have shown that P. agglomerans can also cause walnut bacterial blight (Wang et al., 2016), peach tree canker and Chinese cabbage canker (Guo, 2019), etc. Maize brown stalk rot was first identified in South Africa, caused by Pantoea ananatis and Pantoea sp. of Pantoea, resulting in a serious reduction in maize production in some countries of South African (Goszczynska et al., 2007). At present, this pathogen is mainly distributed in Heilongjiang, Jilin and Liaoning provinces with poplar as its host in China, and has become a major forest disease in Northeast China. Seedlings, young forests and mature forests can occur to varying degrees, and seedlings and young forests are most affected. Poplar bacterial canker belongs to the Quarantine Pests for Import Plants to the People's Republic of China (Yao, 1996, China Agriculture Press, pp.12-27).

In this study, P. agglomerans was first detected on poplar hosts in Shuangshu Nursery, Huzhu County, Qinghai province in Northwest China, causing poplar bacterial canker. After several sampling investigations, it was found that the disease was serious from May to August every year, and the average mortality rate of Populus cathayana in the nursery was 30%, with a maximum of 55% (Fang, 1998, China Agriculture Press, pp.6-416). Poor overall growth and high mortality, resulting in huge economic losses to the seedling cultivation industry. Therefore, this study sampled and investigated the incidence of poplar disease in the Shuangshu Nursery of Huzhu County, and isolated and identified the pathogens. It provides scientific basis for effective prevention and control of poplar diseases in the nursery and further research, and contributes to local ecological construction and environmental protection.

1 Results and Analysis

1.1 Symptoms disease of Populus cathayana

The bark of diseased trees was black and moist. Yellow pith tissue, cracks from pith to bark in severe cases. Pith from the early yellowish to black, brown mucus outflow. The xylem of the branches of the disease is yellowish, extended up and down. From the cross sectional view of the typical specimens (Figure 1A; Figure 1B), both roots and branches occurred, and gradually developed from heartwood to sapwood, presenting a ring structure. Longitudinal section (Figure 1C) showed that the pith is completely black and dead, and the xylem is yellow. After the bark is removed, cracks in the diseased part are clearly visible (Figure 1D). Leaf curl, leaf margin black, water loss, slightly dry phenomenon (Figure 1E). It is speculated that this disease has developed seriously, the xylem up to the branch, down to the root, the whole from inside to outside withered yellow necrosis, leaf wilt, wither, the whole plant died.

Figure 1 Disease symptoms of Poplar cathayan Note: A,B: Cross sectional view; C: Longitudinal section; D: Fracture map of diseased part; E: Leaf morphology

1.2 Pathogenicity detection

A total of 1 strain was isolated from this disease specimen. After repeated culture, observation, selection, morphological identification and molecular fragment sequence analysis, a pure bacterial strain H₁ of Pantoea was obtained, and the pathogenicity was detected by in vitro inoculation. The inoculated branches were placed in an incubator at 28℃ for culture. After 30 days of inoculation, strain H₁ was inoculated (Figure 2A; Figure 2B), yellow pith and black between xylem and phloem, which can be peeled by hand. The symptoms are similar to those in the forest, and the control group has no symptoms (Figure 2C). The reisolation was carried out to obtain the same strain as the tested strain. Again, the pathogenicity of the isolated strain was detected according to Koch postulates, and the results were consistent with the initial test results, in line with Koch postulates, which proved that the isolated strain H₁ was a pathogenic bacterium.

Figure 2 Symptom of Poplar cathayan after artificial inoculation with pathogen Note: A,B: Pathogenicity tests of strain H1; C: Sterile water control

1.3 Morphological identification

Pathogenic bacteria H₁ was cultured in beef extract peptone medium (pH 7.4~pH 7.6) in a constant temperature incubator at 28℃, and then a single colony was obtained by streak plate, usually cultured for 18~48 h. Strain H₁ was grayish white with smooth surface and clear edge at the beginning (Figure 3A), and turned yellow and smelly at the later stage of culture. Strain H₁ was identified as Gram-negative bacteria by Gram staining (Figure 3B). Strain H₁ was rod-shaped under electron microscopy, without periflagella (Figure 3C).

Figure 3 Colony morphology and microscopic examination of strains H1 Note: A: Colony morphology of strain H1; B: Gram stain H (×100)); C: Electron microscopy (sem) image H1

1.4 Molecular biological identification

Primers 27F/1492R (Weisburg et al., 1991) were used for PCR amplification of bacterial 16S rRNA gene. The amplified product was detected by 1% agarose gel electrophoresis and specific fragment H₁ was obtained (Figure 4). After sequencing, the fragment was 1 100 bp, and the homology comparison results showed that the 16S rRNA sequence of strain H₁ was 100% homology with P. agglomerans (GenBank number: CP034148). Strain H₁ was identified as P. agglomerans (GenBank number: MT499447). Then, 16S rRNA phylogenetic tree of H₁ was constructed by the Neighbor-joining method of MEGA 5.2 (Figure 5). Strain H₁ and Pantoea agglomerans (GenBank number: CP034148) clustered on the same branch, and P. agglomerans was further confirmed as the pathogen of poplar bacterial canker by morphological characteristics and molecular biological identification.

Figure 4 Electrophoresis figure of PCR amplified product Note: M: DL2000 DNA Marker; H1: The strainH1; H2: H1 repeat

Figure 5 16S rRNA phylogenetic tree of H1

2 Discussion

The isolation, identification and pathogenicity tests of the pathogens of poplar stem diseases in Shuangshu Nursery, Huzhu County, Qinghai province showed that Pantoea agglomerans was the pathogen of poplar stem disease. Pantoea agglomerans belongs to the genus of Pantoea in Enterobacteriaceae, Enterobacterales, Gammaproteobacteria, causes plant canker. Gavini et al. (1989) established a new genus of Corn fusarium wilt in Erwinia and named it as Pantoea, which is widely found in plant surfaces, soil and animal bodies. Beji et al. (1988) and Gavini et al. (1989) found that Pantoea includes two species P. dispersa and P. agglomerans. Subsequently, E. ananas (Kageyama et al., 1992) and E. stewartii (Zhu et al., 2006, Chinese Journal of Nosocomiology, 16(9): 1006) were also classified as Pantoea. P. agglomerans is a model strain of Pantoea, which can cause plant canker.

Since 1986, Xiang and Shao (1992, Northeast Forestry University Press, pp.119-129) have systematically studied poplar bacterial canker disease in Northeast China. The results showed that after poplar was infected with the disease, elliptic small nodules were formed on the branches in the early stage, and gradually increased, with rough surface and longitudinal cracking, and brown mucus flowed out in summer, with a bad smell. Xylem initially began to change color from the heartwood to the edgewood, showing a ring, and the outermost was white. On beef extract peptone medium, the colony was round, smooth surface, complete edge, gray white. At the later stage of culture, insoluble yellow pigment was produced, and the colony was pale yellow, translucent and slightly sticky. Gram staining was negative, and the flagella was peripherally sparse and curved. Compared with Xiang et al. (2001), in this study, the strain H₁ isolated and purified after being infected with poplar was observed by electron microscopy on the basis of culture characteristics, pathogenicity detection and staining observation, etc. The morphological characteristics of pathogenic bacteria were observed, and no peripheral flagella was observed, indicating slight differences in cell morphology. The 16S RNA sequence of H₁ amplified by PCR further confirmed that H₁ was P. agglomerans of P. gavini.

Xiao et al. (2017) simultaneously isolated P. agglomerans and Xanthomonas arboricola from walnut specimens. P. agglomerans had weaker virulence to walnut leaves and fruits than Xanthomonas arboricola, and there was a compound infection phenomenon. In this study, a large number of pathogenic bacteria H₁ was isolated from the poplar strain with strong pathogenicity and serious harm. It has not been reported in Northwest China that P. agglomerans caused poplar bacterial canker. This study did show that P. agglomerans infected poplar in Shuangshu Nursery, Huzhu County, Qinghai province in northwest China and caused serious poplar bacterial canker. It provides the basis for the prevention and control of poplar stem diseases in the nursery.

3 Materials and Methods

3.1 The situation of sample plot

The Shuangshu Nursery of Huzhu County is one of the state-owned nurseries directly under the jurisdiction of the Forestry Bureau of Huzhu County, with the largest seedling area in Qinghai Province. East longitude 36°44′52.29″, north latitude 101°54′24.29″, altitude 2 430 m, annual precipitation 460 mm, annual average temperature 3.4℃, is a cool temperature climate. P. cathayana is an important economic plant in local nurseries. In recent years, there have been serious diseases in the branches and stems of poplar in the nursery, and it is difficult to cultivate the seedlings. Seedlings, young forests and mature forests were be infected with diseases in varying degrees, causing serious losses to the forestry production in the nursery (Figure 6).

Figure 6 The situation of sample plot Note: A,B: Fracture map of diseased part; C,D: Cross sectional view

3.2 Sample collection, isolation and purification

The typical disease specimens were collected in P. cathayana forest area of Shuangshu Nursery in Huzhu County. The method of collection is to select the infected parts from the diseased plants, cut them with branch shears and saws, take them back to the laboratory, wash them with running water, observe and photograph the symptoms of the victims. Then, the diseased tissues were isolated and cultured by tissue separation method (Fang, 1998, China Agriculture Press, pp.6-416). The pith and the junction of xylem disease were cut into small cubes of about 0.5 cm×0.5 cm, and soaked in 70% alcohol for 15 s. Then the surface was disinfected with 0.1% HgCl₂ for 25 s, rinsed with sterile water for 3 times, dried with sterilized absorbent paper, and cultured in beef extract peptone medium at 28℃. After repeated separation and purification, bacterial strain H₁ was drawn as a single colony by streak plate to obtain the purified strain, which was then stored in a refrigerator at 4℃ for use.

3.3 Morphological identification of pathogenic bacteria

The colony morphology, color and smell of the purified bacterial strain H₁ were observed. Gram staining was performed to determine negative, and scanning electron microscopy was taken to observe the morphological structure.

3.4 Pathogenicity detection of pathogenic bacteria

Koch postulates were used for verification (Fang, 1998, China Agriculture Press, pp.6-416). Healthy four-year-old poplar seedlings were used in cross section by needle injection (Manners, 1979). The isolated bacterial solution of 1×10⁸ cfu/mL (Xiao et al., 2017) was prepared, and the bacterial solution was injected into the pith of poplar and the junction with xylem with sterile syringe, and sterile water was dropped every 6 h for constant temperature culture. Each strain was repeated 3 times and inoculated with sterile water as CK. The inoculated branches were placed in an incubator at 28 ℃ for culture.

3.5 Molecular biological identification of pathogenic bacteria

All the bacterial pathogens H₁ were negative by Gram staining, and then DNA extraction of two bacterial strains was carried out: single colonies were selected and placed in a centrifuge tube filled with 1 mL sterile double distilled water, centrifuged at 12 000 r/min for 2 min, and the supernatant was discarded. Then, by boiling method (G€ssow and Tim, 1989), 500 μL sterile double distilled water was added to the centrifuge tube, and the centrifuge was placed in 92℃ water bath for 5 min and centrifuged at 12 000 r/min for 2 min. The above clear liquid was used as a template. The PCR primer pairs were 27F (5‘-AGAGTTTGATCCTGGCTCAG-3’) and 1492R (5’-TACGACTTAACCCCAATCGC-3’). PCR amplification procedure: pre-denaturation at 94℃ for 5 min; In the PCR cycle, 94℃ for 30 s, 52℃ for 45 s, 72℃ for 1 min, 30 cycles. Finally, extension at 72℃ for 8 min (Li, 2013). The above PCR products were detected by 10 g/L agarose gel electrophoresis with Goldview, and the effect of genomic DNA extraction of isolated pathogenic bacteria was analyzed according to the electrophoresis pattern. To verify the stability of the method, the number of DNA extraction and PCR amplification tests was appropriately increased, and each test was repeated 3 times. The amplified products were then sent to the sequencing company (Shanghai Sangon Biotech) for sequencing. Blast search was performed on GenBank for sequences with high similarity, and homology comparison was performed.

Authors’ contributions

BLC was the designer and director of this experimental study. XWLT and YGJ participated in the design of the experiment; AXZ participated in sample collection; As the executor of this experiment, BSC carried out disease investigation, sample collection, isolation and identification of pathogens, and finally wrote the paper. All authors read and approved the final manuscript.

Acknowledgements

This study was supported by the Science and Technology Cooperation Program for Youth (2020-QY-209) of Qinghai Provincial Science and Technology Department.

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