Pseudomonas synxantha for preventing and treating botrytis cinerea and prolonging the flowering period of Chinese rose and its application

Abstract

The application discloses a strain of Pantoea dispersa for preventing and treating Chinese rose gray mold and prolonging the flowering period of Chinese rose, and application thereof. The strain is Pantoea dispersa SJJ17CGMCC NO.30658. The strain can antagonize plant pathogens such as Colletotrichum gloeosporioides, Alternaria solani, Fusarium proliferatum and Fusarium oxysporum. The antagonistic substance produced by the strain has certain resistance to high temperature and ultraviolet rays. The strain can also produce siderophores and has the characteristics of dissolving inorganic phosphorus. The application provides an excellent biocontrol strain for preventing and treating Chinese rose gray mold, and has great production significance for green prevention and control technology of Chinese rose diseases and sustainable development of the Chinese rose industry.

Description

TECHNICAL FIELD

[0001] The present application relates to a kind of dispersed panus with antagonism to rose gray mold fungus and its application, in particular to a kind of dissolved phosphorus, produces siderophore dispersed panus SJJ17 with antagonism to rose gray mold fungus and its application in rose gray mold disease prevention, flowering period extension and growth promotion. BACKGROUND

[0002] Rose is the common name of hybrid varieties group of Rosa plant after hybridization, not only one of the ten traditional Chinese famous flowers, one of Beijing flowers, also has the reputation of "Queen of Flowers", is one of the important flowers in daily life. In recent years, with the expansion of rose planting area and planting area year by year, the economic value of cut flowers is rising year by year, and the good prospect, improving the quality of rose has become the focus of the industry at present. And in the growth period of rose, it is always invaded by gray mold, anthracnose, powdery mildew, black spot, downy mildew and other diseases, which not only reduces the ornamental value of rose, but also seriously affects the growth of plant. Therefore, strengthening the prevention and control of rose disease is the key to ensure the healthy development of rose industry.

[0003] Gray mold is a common plant disease, and its pathogen is Botrytis, which can occur in many plants such as rose, tomato and grape. Botrytis cinerea can infect host in the form of mycelium, conidium and sclerotium, and the infection process and mechanism are very complex, with multiple infection modes. When Botrytis cinerea infects rose, it mainly infects flowers, which can cause petal browning, rotting, wilting and even whole flower browning and wilting, thereby reducing the ornamental value of flowers. In recent years, with the increasing economic value of rose, flower farmers have adopted continuous cropping mode to plant cut rose, which not only brings high income but also leads to serious occurrence of gray mold. Gray mold is very harmful to rose. Once it occurs, even the rose with light symptoms cannot be sold due to damaged petals, which greatly reduces its economic value. At present, the prevention and control methods of rose gray mold at home and abroad are single, mainly chemical control, such as using carbendazim and prochloraz. Long-term use of large amount of chemical agents not only increases cost but also pollutes environment. In addition, the use of chemical agents can also cause directional selection of pathogenic bacteria and produce drug resistance. Therefore, the application of green and pollution-free biological control to prevent and control rose gray mold is the direction of future development. SUMMARY

[0004] The purpose of the present application is to provide a strain of bacteria with antagonism to rose gray mold fungus, which can dissolve phosphorus and produce siderophore, and its application in rose gray mold disease prevention, growth promotion and flowering period extension.

[0005] The rose gray mold disease prevention, flowering period extension and growth promoting bacteria provided by the present application are Pantoeadispersa SJJ17.

[0006] The Pantoea dispersa SJJ17 has been preserved in the China General Microbiological Culture Collection Center (CGMCC) on May 16, 2024, and the preservation number is CGMCC No. 30658. The Pantoea dispersa SJJ17 is a gram-negative bacterium isolated from rose leaves. The bacterium can form a yellowish colony on LB medium, and the colony is round, smooth, flat and opaque, with a smooth surface.

[0007] The bioinoculant or microbial fertilizer containing the above-mentioned Pantoea dispersa SJJ17 as an active ingredient also belongs to the protection scope of the present application.

[0008] Experiments prove that the bacterium having antagonistic effect on the rose gray mold pathogen in the present application is the Pantoea dispersa SJJ17, which can effectively prevent and control the rose gray mold, and can effectively promote the growth of rose cutting seedlings and prolong the flowering period of roses.

[0009] The bacterium having growth-promoting and disease-preventing effects on roses in the present application is the Pantoea dispersa SJJ17, which is isolated from rose leaves and is an endophytic bacterium of roses. The plate confrontation experiment shows that the strain has good antagonistic effect on the rose gray mold pathogen Botrytis cinerea, the garden plant pathogen Glomerella cingulata, Alternaria solani, Fusarium proliferatum and Fusarium oxysporum. The strain can also produce siderophores and dissolve inorganic phosphorus. The in vitro petal protection test shows that the strain has a 100% control effect on the rose gray mold. The greenhouse experiment shows that the strain can prolong the flowering period by 1.9 days. The plant height, aboveground fresh weight and leaf number of the rose cutting seedlings treated with the strain are significantly higher than those of the control. The acquisition of the strain is expected to provide an environmentally friendly, simple and effective way for disease control and growth promotion of roses. The Pantoea dispersa SJJ17 of the present application is a strain with good disease-preventing and growth-promoting application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1 The results of the confrontation culture test of the Pantoea dispersa SJJ17 and the rose gray mold fungus.

[0011] Figure 2 The results of the confrontation culture test of SJJ17 and other plant pathogenic fungi.

[0012] Figure 3The results of SJJ17 bacteria and metabolic liquid in preventing and controlling the gray mold of Rosa chinensis 'Nihangpao' and 'Carola' were determined by petal method.

[0013] Figure 4 The results of SJJ17 bacteria suspension treatment in promoting the growth of Rosa chinensis cutting seedlings were determined.

[0014] Figure 5 The preservation effect of SJJ17 treatment on bottle cutting 'Carola' cut flowers was determined.

[0015] Figure 6 The transparent circle formed around SJJ17 bacterial colonies on siderophore and dissolved inorganic phosphorus detection plates.

[0016] Biological material preservation

[0017] Preservation number: CGMCC No. 30658

[0018] Name: Pantoea dispersa SJJ17

[0019] Classification name: Pantoea dispersa

[0020] Whether alive: alive

[0021] Preservation time: May 16, 2024

[0022] Preservation agency: China General Microbiological Culture Collection Center, Beijing Chaoyang District, No. 1, Xibei Road, No. 3 DETAILED DESCRIPTION

[0023] The methods in the following examples are all conventional methods unless otherwise specified.

[0024] Example 1, screening of antagonistic bacteria against Rosa chinensis gray mold

[0025] This strain was isolated from Rosa chinensis leaves planted on the campus of Beijing Agricultural University for more than 10 years. The specific method is as follows: 5 g of healthy Rosa chinensis leaves were first washed with clean water, and then treated with 75% ethanol for 30 s, and then washed with sterile water for 3 times. After sterile grinding, 10 mL of sterile water was added, and the mixture was placed in a shaking bed at 170 rpm for 30 min, and then left to stand until obvious supernatant appeared; the supernatant was diluted by 10 times in sterile water, and then 10 -3100 μL of each dilution was uniformly plated on LB (yeast extract 5 g, tryptone 10 g, sodium chloride 10 g, distilled water to 1000 ml, pH 7.0) medium, inverted and placed in a 28°C incubator in the dark for 24 h. After single colonies appeared, well-grown colonies with different color and morphology were immediately picked and purified for preservation. Fresh rose gray mold pathogen (isolated from rose gray mold disease plants and stored in the College of Landscape Architecture, Beijing Agricultural University) was inoculated in the center of the inverted PDA plate (potato 200 g, sucrose 20 g, agar 20 g, distilled water 1000 ml), and the purified strain was inoculated on the sterile toothpick 3 cm away from the bacterial cake. After inoculation, the plates were incubated at 28°C in the dark, and the production of inhibition zones was observed to screen for antagonistic strains. A total of 5 strains with antagonistic effects on rose gray mold pathogen were screened, of which the best one was named SJJ17. The antagonistic effect of the strain on rose gray mold pathogen is shown in Fig. 1. Figure 1

[0026] Example 2, Species identification of SJJ17 strain

[0027] The SJJ17 strain was identified by routine physiological and biochemical tests and 16S rDNA sequence analysis, and it was confirmed that the strain belongs to Pantoea dispersa. The physiological and biochemical characteristics and basic biological characteristics of the strain are shown in Table 1.

[0028] Table 1. Basic biological characteristics of SJJ17

[0029]

[0030] Note: "+" indicates that the carbon source (or nitrogen source) can be utilized, and "-" indicates that the carbon source (or nitrogen source) cannot be utilized

[0031] The genomic DNA of Pantoea dispersa SJJ17 was used as a template, and bacterial 16S rDNA sequence universal primers were used as primers (27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; reverse primer 1492R: 5'-TACGGGTACCTTGTTACGACTT-3'), and a product fragment of about 1.5 kb was amplified by PCR. 1368 nucleotides were obtained by sequencing with 27F primer (Sequence 1 in the sequence listing). The sequence was compared in the NCBI database, and a phylogenetic tree of SJJ17 and related bacteria was constructed using MEGA 7.0.21 software. The results showed that the 16S rDNA sequence of SJJ17 had the highest homology with the 16S rDNA sequence of Pantoeadispersa BJQ0007 (GenBank: CP045216.1), reaching 99.93%. ​

[0032] According to the Handbook of Common Bacterial Identification, the physiological and biochemical characteristics of SJJ17 strain are the same as those of Pantoea dispersa. Combined with the above molecular identification, it is identified as Pantoea dispersa. SJJ17 is named as Pantoea dispersa SJJ17. The Pantoea dispersa SJJ17 strain has been preserved in the China General Microbiological Culture Collection Center (CGMCC, located at No. 1, Yihuangyuan, Beichen West Road, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences) on May 16, 2024, and the preservation number is CGMCC NO. 30658.

[0033] Example 3, antagonistic effect of Pantoea dispersa SJJ17 on other garden plant pathogens

[0034] Strain SJJ17 was inoculated into a 250 mL conical flask containing 100 mL of LB liquid medium, and cultured at 28°C, 180 rpm in the dark for 48 h. The bacterial solution was diluted with sterile water to a concentration of 1×10 7 CFU / mL. The plant pathogenic Colletotrichum gloeosporioides, Alternaria solani, Fusarium proliferatum and Fusarium oxysporum were cultured on PDA plates for 7 days at 28°C in the dark. A 0.5 cm diameter punch was used to take a bacterial cake from the edge of the colony and placed in the center of a new PDA medium. 2 μL of the prepared SJJ17 bacterial suspension was inoculated at a distance of 3 cm from the center of the bacterial cake, 3 points per dish. After the bacterial suspension was dried, it was cultured at 28°C in the dark for 7 days. The results showed that strain SJJ17 could antagonize Colletotrichum gloeosporioides, Alternaria solani, Fusarium proliferatum and Fusarium oxysporum Figure 2 ).

[0035] Example 4, determination of the control effect of Pantoea dispersa SJJ17 on gray mold of rose

[0036] The spores of PDA plate cultured for 7 days of the gray mold pathogen Botrytis cinerea were washed with 0.01% Tween, filtered with double-layer gauze, and then adjusted to a concentration of 1×10 5 / mL with sterile water for standby. Strain SJJ17 was cultured overnight according to the method in Example 3, and then centrifuged at 5000 rpm for 10 min to collect the bacterial cells and supernatant. The bacterial cells were adjusted to 1×10 7 CFU / mL with sterile water. Healthy petals of the rose variety ‘Nihongpao’ were selected, disinfected with 75% alcohol for 30 s, washed with sterile water for 3 times, and then dried the water stains on the super-clean bench. The petals were placed on 0.4% water agar medium. 20 μL of the prepared SJJ17 bacterial suspension or supernatant was inoculated on each petal, and then dried and inoculated with 1×10 5 / mL of Botrytis cinerea spore suspension, dried and incubated at 22℃ for 12 / 12h light / dark. Meanwhile, healthy petals of Rosa hybrida cultivar 'Carola' were sterilized and punched into 2.8cm diameter discs with a puncher, inoculated with B. cinerea and SJJ17 according to the above method. Each treatment was replicated three times, and sterile water was used as control. After 3 days of incubation, the lesion area was measured and the control efficiency was calculated according to the following formula: Control efficiency % = [(lesion area of control - lesion area of treatment) / lesion area of control] x 100%.

[0037] The results showed that after 3 days of inoculation with B. cinerea, the lesion area almost covered the whole petal of 'Nihongpao', the petal changed from pink to yellowish brown, and the mycelium of the pathogen appeared on the lesion area. Figure 3 However, the petals pre-inoculated with SJJ17 metabolites and cells were completely free of disease, with a control efficiency of 100% (Table 2). Similarly, yellow lesions appeared on the petals of 'Carola' after 3 days of inoculation with B. cinerea, while the lesion area of petals pre-inoculated with SJJ17 metabolites and cells was significantly reduced Figure 3 , with control efficiencies of 83.3% and 73.8%, respectively (Table 2).

[0038] Table 2. Control efficiency of SJJ17 against B. cinerea on detached petals of Rosa hybrida

[0039]

[0040] Note: Different letters in the same column indicate significant differences (P < 0.05).

[0041] Example 5. Effect of Pantoea dispersa SJJ17 on the growth of Rosa hybrida cutting seedlings

[0042] Healthy Rosa hybrida 'Yueyuefen' branches with similar thickness and length were selected to prepare cuttings, with 2 nodes per cutting. The cuttings were planted in pots filled with perlite, peat and vermiculite (1:1:1). SJJ17 bacterial suspension with a concentration of 1 x 10 7 CFU / mL was prepared according to the method in Example 3. 100 mL of the prepared bacterial suspension was poured at the base of the cutting seedlings. Each treatment had 5 seedlings, with 4 replicates, and sterile water was used as control. The treated cuttings were incubated at 24℃ for 16h / 8h light / dark. The growth of the cuttings was investigated after 30 days of incubation. The results showed that the plant height, fresh weight of aboveground part and leaf number of the cuttings treated with SJJ17 bacterial suspension were significantly higher than those of the control (Table 3 and Figure 4 ). These results indicated that SJJ17 could promote the growth of the aboveground part of Rosa hybrida cutting seedlings.

[0043] Table 3, Effect of SJJ17 on the growth of cuttings

[0044] Growth index Sterile water control SJJ7 bacterial suspension treatment Plant height (mm) 68.5±3.4 90.5 ± 3.3 * ]]> Fresh weight of aboveground part (g) 0.38±0.03 1.25 ± 0.14 * ]] Leaf number 6.7±0.9 13.9 ± 0.9 * ]]

[0045] * The treatment group and the control group were significantly different (P < 0.05).

[0046] Example 6, Determination of Pantoea dispersa SJJ17 to extend the vase life of cut flowers

[0047] SJJ17 bacterial suspension with a concentration of 1 x 10 7 CFU / mL was prepared according to the method in Example 3. The cut flowers 'Carola' with full flower heads, fresh degree, and basically consistent opening degree were used as materials, and the treatments were carried out according to Table 4. There were 3 cut flowers per treatment and 5 replicates. The flowering index and its duration of each flower were recorded daily during the vase period. The grading standard of flowering index: 0 level, calyx upright; 1 level, calyx horizontal; 2 level, calyx drooping, outer petals begin to loosen; 3 level, initial opening, outer petals unfolded; 4 level, full bloom, multiple layers of petals unfolded; 5 level, full bloom, flower exposed; 6 level, petals turned over or began to wilt. The vase life refers to the number of days from the day of vase to the day before the appearance of wilting, neck bending, or bluing, etc. (i.e. flowering index 6 level).

[0048] The results showed that the vase life of the control group was only 8.6d, after which the flower branches appeared neck bending, drooping, obvious chlorosis of leaves, severe wilting and other phenomena. However, the use of SJJ17 bacterial suspension to spray the flower buds could significantly prolong the vase life of the rose to 10.5d, 1.9d longer than the sterile water control group (Table 4). Figure 5 These results showed that SJJ17 could be used for the preservation of fresh cut roses.

[0049] Table 4, Preservation effect of SJJ17 treated vase 'Carola' cut flowers

[0050]

[0051]

[0052] Note: Different letters in the same column indicate significant difference (P < 0.05).

[0053] Example 7, Pantoea dispersa SJJ17 can dissolve inorganic phosphorus

[0054] The activated SJJ17 was inoculated into LB liquid medium and shaken overnight at 28°C, 200 rpm. 10 μL of the culture was inoculated onto inorganic phosphorus medium plates (yeast powder 0.5 g, glucose 10 g, tricalcium phosphate 5 g, ammonium sulfate 0.5 g, magnesium sulfate 0.3 g, potassium chloride 0.3 g, sodium chloride 0.3 g, manganese sulfate 0.03 g, ferrous sulfate 0.03 g, agar 15 g, distilled water 1000 mL) and repeated 3 times. After drying in a clean bench, the plates were cultured at 28°C in the dark for 7 days, and the appearance of a hydrolysis halo around the colonies was observed. The results showed that a hydrolysis halo appeared around the colonies of SJJ17 on the inorganic phosphorus medium plates Figure 6 ), indicating that SJJ17 can hydrolyze inorganic phosphorus.

[0055] Example 8, Pantoea dispersa SJJ17 can produce siderophores

[0056] 10 μL of the culture was inoculated onto siderophore-CAS agar medium plates (Chrome Azurol S 60.5 mg, cetyltrimethylammonium bromide 72.9 mg, ferric chloride hexahydrate 2.645 mg, sodium phosphate monobasic dihydrate 295.25 mg, sodium phosphate dibasic dodecahydrate 1213.5 mg, ammonium chloride 125 mg, potassium phosphate dibasic 37.5 mg, sodium chloride 62.5 mg, agar 9 g, distilled water to 1000 mL, pH 6.8 ± 0.2) and repeated 3 times. After drying in a clean bench, the plates were cultured at 28°C in the dark for 3 days, and the color change of the medium around the colonies from blue to orange was observed. The results showed that a blue discoloration halo appeared around the colonies of SJJ17 Figure 6 ), indicating that SJJ17 has the ability to produce siderophores.

[0057] Example 9, determination of the stability of the antagonistic substances produced by Pantoea dispersa SJJ17 to temperature and ultraviolet light

[0058] The method of Example 3 was used to prepare 1 x 10 7SJJ17 bacterial suspension. 1 mL of prepared SJJ17 bacterial suspension was transferred into a 250 mL flask containing 100 mL of LB liquid medium, and incubated at 28°C, 180 rpm for 7 days. After centrifugation at 12000 rpm for 10 min, the supernatant was obtained by filtration with a microporous membrane. To determine the stability of SJJ17 metabolites to temperature, the prepared supernatant was treated at 4, 30, 60, 80 and 121°C for 20 min, respectively. To determine the stability of SJJ17 metabolites to UV irradiation, the prepared supernatant was irradiated at a distance of 30 cm from a UV lamp (TL20W / 12RS, Philips) for 5, 15, 25, 35 and 45 min, respectively. The P. rosae was cultured according to the method of Example 1, and a 0.5 cm diameter P. rosae mycelium was placed in the center of PDA medium. Sterile filter paper pieces were placed at a distance of 3 cm from the center of the plate, and 3 pieces were placed in each dish. 2 μL of the sterile supernatant of SJJ117 treated at different temperatures and UV irradiation times was added to the filter paper pieces. Each treatment was repeated 3 times, and the untreated supernatant was used as a control. After incubation at 28°C for 7 days, the radius of P. rosae was measured, and the inhibition rate was calculated according to the formula:

[0059] Inhibition rate = (colony diameter of control group - colony diameter of treatment group) / (colony diameter of control group - mycelium diameter) x 100%

[0060] The results showed that there was no difference in the inhibition activity of the sterile supernatant of SJJ17 treated at 4°C and 30°C compared with the control group, while the inhibition rate of the sterile supernatant of SJJ17 treated at 60°C and 80°C decreased to 43.6% and 44.7%, respectively. The sterile supernatant of SJJ17 treated at 121°C for 20 min had no inhibition effect (Table 5). The inhibition rate of the sterile supernatant of SJJ17 treated with UV irradiation for 5 min decreased to 57.2%, while there was no difference in the inhibition rate of the sterile supernatant of SJJ17 treated with UV irradiation for 15, 25 and 35 min, which was 42.2%-45.4%. The inhibition rate of the sterile supernatant of SJJ17 treated with UV irradiation for 45 min was 38.1% (Table 6). These results indicated that the inhibition substance produced by SJJ17 was stable to temperature and UV irradiation.

[0061] Table 5, determination of the stability of SJJ17 metabolites to temperature

[0062] Treatment temperature (°C) Bacteriostatic rate (%) CK 64.8±1.5a 4 57.1±2.1a 30 65.6±5.8a 60 43.6±1.3b 80 44.7±1.5b 121 0.0±0.3c

[0063] Table 6, determination of the stability of SJJ17 metabolites to UV irradiation

[0064]

[0065]

Claims

1. A strain of Pantoea dispersa, characterized in that, Pantoea dispersa (Pantoea dispersa) Pantoea dispersa ) SJJ17, having a preservation number of CGMCC No. 30658 at the China General Microbiological Culture Collection Center.

2. Use of Pantoea dispersa as claimed in claim 1 for controlling plant diseases; the plant disease being grey mould of rose caused by Botrytis cinerea.

3. Use according to claim 2, characterized in that, the plant disease being a disease occurring on rose.

4. The use of the Pseudomonas dispersa of claim 1 in the preparation of a biocontrol agent or microbial fertilizer against one or more of the plant pathogenic fungi Glomerella cingulata (G. cingulata) Colletotrichum gloeosporioides , Alternaria solani (A. solani) Alternaria solani , Fusarium proliferatum (F. proliferatum) Fusarium proliferatum and Fusarium oxysporum (F. oxysporum) Fusarium oxysporum .​​​​​​​​​ 5. A biocontrol agent for controlling plant diseases, characterized by comprising the bacteria of claim 1. The active ingredient of the biocontrol agent is Pantoea dispersa (CGMCC No. 30658) Pantoea dispersa ) SJJ17, and the plant disease is grape gray mold caused by Botrytis cinerea.

6. A microbial inoculant for controlling plant diseases, characterized by comprising the microorganism according to any one of claims 1 to 5. The active ingredient of the microbial fertilizer is Pantoea dispersa (Pantoea agglomerans) Pantoea dispersa ) SJJ17, the preservation number of the Pantoea dispersa is CGMCC No. 30658; and the plant disease is the gray mold of Chinese rose caused by Botrytis cinerea.

7. Use of Pantoea dispersa as claimed in claim 1 for solubilizing phosphorus and producing siderophores.

8. Use of Pantoea dispersa as claimed in claim 1 for promoting growth of rose and prolonging the vase life of rose cut flowers.

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