Combined microbial agent for preventing and treating nematodes and application of combined microbial agent in prevention and treatment of pepper root-knot nematodes

Abstract

The invention discloses a combined microbial agent for preventing and treating nematodes and application of the combined microbial agent in prevention and treatment of pepper root-knot nematodes. The combined microbial agent comprises the following active components: 1) streptomyces WZSF-1 or a fermentation supernatant thereof, and 2) paecilomyces lilacinus E16 or a fermentation supernatant thereof. The Streptomyces griseorubens WZSF-1 is named as Streptomyces griseorubens WZSF-1, the Streptomyces griseorubens WZSF-1 is classified and named as Streptomyces griseorubens, and the Streptomyces griseorubens WZSF-1 has a preservation number of GDMCC (Microbial Culture Collection Center) No.65368 in Guangdong Province. The Streptomyces griseorubens WZSF-1 is named as Streptomyces griseorubens WZSF-1. Pot experiment results show that E16, WZSF-1 and the compound liquid thereof have promotion effects on plant height, stem diameter, chlorophyll content, overground part fresh weight, root weight and defensive enzyme activity of pepper. Compared with single use of E16 and WZSF-1, the compound treatment has the advantages that the prevention effects are improved by 85.71% and 62.50%.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a combined bacterial agent for preventing and controlling nematodes and application thereof in preventing and controlling pepper root-knot nematodes. Background Art

[0002] Meloidogyne spp. have a wide host range, and most crops are susceptible to them, including cash crops, food crops, fruits and vegetables, horticultural plants, and weeds (Jones et al. 2013, Subramanian et al. 2017). To date, over 100 species of root-knot nematodes have been reported both domestically and internationally, with over 40 currently occurring in my country. The vast majority of plant root-knot nematode diseases are caused by southern, northern, javanic, and peanut root-knot nematodes, with southern root-knot nematodes being the predominant species. The diversity of root-knot nematode species is still evolving, with new species constantly emerging (Liu Weizhi and Duan Yuxi 2000, Yuan Li 2021, Gong Yuanfu 2020). The elephant-ear bean root-knot nematode (Meloidogyne enterolobi) is a recently emerging plant root-knot nematode. First discovered on elephant-ear bean trees in Hainan, it has now been widely reported worldwide. It has strong pathogenicity, a wide host range, high reproductive capacity, and possesses resistance genes to Mi, N, and Rk. It is currently widespread in Hainan and Fujian, my country (Yan Xiaoning et al. 2007, Kiewnick et al. 2008, Elling 2013, Chen Hui et al. 2016). Investigations have shown that the elephant-ear bean root-knot nematode is already widespread in Hainan and can parasitize some of Hainan's major cultivated crops, including cucurbits, southern medicinal plants, solanaceous crops, and vegetables. In recent years, the nematode has been found on pepper roots in Yunnan and Hunan provinces, as well as on cabbage roots in Shaanxi Province, with a trend of gradual expansion from south to north (Zhuo Kan et al. 2008, Jia Benkai et al. 2012, Li Zhourong et al. 2020).

[0003] The root-knot nematode's reproductive life cycle consists of three stages: egg, larva, and adult. Second-stage juveniles (J2) are the only infective stage. They can penetrate the host root's apical growth point, penetrate the intercellular spaces, and reach the vascular parenchyma tissue in the cell division zone to feed (Zhang Chuangong et al. 2014). After colonizing the root system, J2s undergo three molts before emerging as adults. Females remain in the host root system, while males leave. At the end of their life cycle, female egg sacs are shed into the soil, marking the next stage of the infection cycle (Duan Yuxi 2011). Nematodes overwinter in the soil as eggs, accompanying diseased host remains, or as J2s. The following year, when conditions are favorable, the eggs hatch into larvae, and the second-stage juveniles continue their development. Current methods for controlling root-knot nematodes include chemical, physical, biological, agricultural, plant quarantine, and the use of resistant varieties (Lu Xuejun 2010, Hu Yujin 2019).

[0004] Paecilomyces lilacinus is an important biocontrol fungus for controlling several plant diseases. Besides parasitizing nematode eggs and cysts, it can also parasitize female nematodes and larvae. Furthermore, its metabolites exhibit strong nematicidal activity against nematodes, inhibiting the hatching of root-knot nematode eggs and the activity of larvae, thereby reducing nematode damage to crops. This mechanism of action is related to the production of secondary metabolites with nematicidal activity (Sun Manhong et al. 2004, Liu Chunxiu et al. 2005, Sharma et al. 2016, Liu et al. 2022). Sun Manhong treated second-instar larvae of soybean cyst nematodes with the fermentation filtrate of Paecilomyces lilacinus M-14 and found that the filtrate had a strong inhibitory and killing effect against nematodes. Toxicity analysis revealed that the component is a protease (Sun Manhong et al. 2002). Sharma's research found that the culture filtrate of Paecilomyces purpurogenum 6029 had a 100% mortality rate against the second-instar larvae of southern root-knot nematodes. After further cultivation for 15 days, it was found that its fermentation filtrate was more toxic to nematodes (Sharma A et al 2014).

[0005] Actinomycetes primarily control root-knot nematodes through antagonism or toxicity. Nematicidal bacteria are primarily concentrated in the genus Streptomyces. Abamectin, a highly effective nematicidal agent, was isolated from Streptomyces avermitilis and has been widely used as a nematicide, insecticide, and acaricide on fruits, vegetables, and grain crops. Ruanpanun (2010) isolated 83 strains whose metabolites exhibited nematicidal activity from soil samples in Thailand, 97.6% of which were Streptomyces. Jin Han et al. (2023) incubated the fermentation supernatant of Streptomyces nigricans with suspensions of second-instar larvae and eggs of root-knot nematodes to determine their toxic and inhibitory effects on root-knot nematode J2s and nematode eggs. The results showed that after 24 hours of incubation, the supernatant's insecticidal activity against J2s reached 91.21%, and after 72 hours of incubation, the supernatant's inhibitory rate against nematode eggs reached over 80%. Ruanpanun and Chamswarng (2016) isolated 25 actinomycetes from commercial earthworm castings, of which 12 strains had significant inhibitory effects on the eggs and larvae of southern root-knot nematodes. After identification, these 12 actinomycetes were all Streptomyces.

[0006] In practical applications, the application of a single bacterial strain can result in unstable efficacy and a narrow range of pathogen control. This can be due to a variety of factors, including the difficulty of a single bacterial strain adapting to the complex field environment and becoming dominant. Soil is a key factor (Wang Yanan et al. 2022). Studies have found that most beneficial bacteria exhibit synergistic effects on plant growth, with composite biocontrol agents exhibiting advantages over single strains in terms of environmental adaptation and efficacy stability. The use of composite biocontrol agents can enhance or stabilize disease control efficacy (Liu Xin 2022). Related research, both domestically and internationally, has demonstrated that the combined use of different biocontrol agents can alleviate the environmental dependence and high dosage requirements of a single strain, emphasizing the importance of combining different strains to address the shortcomings of a single strain (Rabie 1998, Neeraj and Singh 2011, Mao et al. 1998).

[0007] AM fungi and plant growth-promoting rhizobacteria (PGPR) have great potential in the biocontrol of soil-borne diseases. The combined application of these two strains significantly enhanced tomato growth promotion and inhibited root-knot nematodes (Liu et al. 2012). Castillo et al. (2013) used Paecilomycops lilacinus 251 and Bacillus firmus GB-126 against cotton reniform nematodes. In both laboratory and field experiments, the combined use of these two strains significantly reduced nematode populations. Giri et al. (2022) co-inoculated peppers infected with root-knot nematodes with both arbuscular mycorrhizal fungi and Paecilomycops lilacinus. Compared with either inoculation alone, the dual inoculation significantly promoted pepper growth and reduced the parasitism rate, number of root knots produced, and egg abundance of root-knot nematodes. Paecilomycops lilacinus is widely used in the biocontrol of root-knot nematodes. In practical applications, combining Paecilomycops lilacinus with other biocontrol strains is more effective. Khan et al. (2006) applied a combination of Paecilomyces lilacinus and the nematode-feeding fungus Acremonium truncatum, which reduced the density of root-knot nematodes in the soil by 94% and the number of root knots in the root system by 62%.

[0008] The choice of biocontrol bacteria and the method of compounding them are not arbitrary decisions, and the effectiveness of a mixed strain is not necessarily greater than that of a single strain. For example, Barbosa et al. (2018) found that when two different Bacillus species were mixed together, their effectiveness against sisal stem rot was actually less than when used alone. Therefore, the effects of bacterial strains with strong nematicidal activity on the growth and development of Paecilomyces lilacinus E16 were analyzed using plate confrontation and potency assays. Their compatibility was evaluated, and the ratio of the selected nematicidal actinomycete strains to be compounded with Paecilomyces lilacinus E16 was determined. The effectiveness of the combined strains for controlling root-knot nematodes in potted plant experiments was also analyzed. Summary of the Invention

[0009] Root-knot nematode disease is a global, soil-borne plant disease that causes significant economic losses annually. Traditional nematode control methods are detrimental to sustainable agricultural production due to high costs, toxicity, nematode resistance, and ecological damage. Biological control is currently a hot topic in crop disease prevention and control, but single bacterial strains often exhibit inconsistent efficacy against root-knot nematodes. Composite biocontrol agents offer advantages over single strains in terms of environmental adaptability and efficacy stability, and their use can enhance or stabilize disease control efficacy.

[0010] Based on this, the present invention combines the screened actinomycete strain with high nematicidal activity with the Paecilomyces lilacinus E16 strain to evaluate the control effects of the two biocontrol bacteria and their combination on pepper root-knot nematodes.

[0011] The specific technical solutions are as follows:

[0012] The present invention provides a combined bacterial agent for controlling nematodes, the active ingredients of which are composed of the following components:

[0013] 1) Streptomyces WZSF-1 or its fermentation supernatant,

[0014] 2) Paecilomyces lilacinus E16 or its fermentation supernatant,

[0015] The Streptomyces WZSF-1 is named as Streptomyces griseorubens WZSF-1, classified as Streptomyces griseorubens, and its preservation number in Guangdong Provincial Microbiological Culture Collection Center is GDMCC No. 65368.

[0016] Preferably, the volume ratio of the fermentation supernatant of Streptomyces WZSF-1 to the fermentation supernatant of Paecilomyces lilacinus E16 is 4:1.

[0017] The preparation method of the Streptomyces WZSF-1 fermentation supernatant is as follows:

[0018] 1) Preparation of Streptomyces WZSF-1 seed solution

[0019] A single colony was picked from the activated Streptomyces WZSF-1 plate with a sterile toothpick and cultured in 100 mL YE medium at 28°C with shaking for 3 days to obtain the Streptomyces WZSF-1 seed solution;

[0020] 2) Preparation of fermentation supernatant

[0021] The seed liquid of Streptomyces WZSF-1 was inoculated into Gao's No. 1 liquid culture medium at a volume percentage of 1%, and cultured at 28°C for 7 days. The fermentation liquid of each strain was centrifuged at 10,000 r / min for 10 minutes to obtain the fermentation supernatant of Streptomyces WZSF-1.

[0022] The preparation method of the fermentation supernatant of Paecilomyces lilacinus E16 is as follows:

[0023] 1) Preparation of Paecilomyces lilacinus E16 seed solution

[0024] A single colony of Paecilomyces lilacinus E16 was picked and cultured in 100 mL of PDB medium at 28°C for 3 days to obtain the seed solution of Paecilomyces lilacinus E16;

[0025] 2) Preparation of fermentation supernatant

[0026] The seed liquid of Paecilomyces lilacinus E16 was inoculated into PDA liquid culture medium at a volume percentage of 1%, and cultured with shaking at 28° C. for 7 days. The fermentation liquid of each strain was centrifuged at 10,000 rpm for 10 minutes to obtain the fermentation supernatant of Paecilomyces lilacinus E16.

[0027] The application of the combined bacterial agent in the prevention and treatment of root-knot nematodes also falls within the scope of protection of the present invention.

[0028] Preferably, the root-knot nematodes are root-knot nematodes of the elephant ear bean family and root-knot nematodes of the southern region.

[0029] Beneficial effects of the present invention:

[0030] 1. Determination of the antagonism and combined activity of Streptomyces WZSF-1 and Paecilomyces lilacinus E16: The antagonism between WZSF-1 and Paecilomyces lilacinus E16 was evaluated by the plate antagonism method, and the results showed that the antagonism rate between the two strains was small; after the fermentation supernatants of the two strains were compounded in different proportions, their nematicidal activity was measured, and it was found that when the ratio of E16 to WZSF-1 was 1:4, its nematicidal activity increased, which was 8.28% and 1.57% higher than when E16 and WZSF-1 were used alone, respectively.

[0031] 2. Potted Plant Control Efficacy Test: Results from potted plant tests showed that E16, WZSF-1, and their combined solution promoted plant height, stem diameter, chlorophyll content, aboveground fresh weight, root weight, and defensive enzyme activity in peppers. Both strains and their combined solution demonstrated control efficacy against root-knot nematodes, with efficacy rates of 43.75%, 50.00%, and 81.25%, respectively. Compared to using E16 and WZSF-1 alone, the combined treatment increased control efficacy by 85.71% and 62.50%, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The culture characteristics of strain WZSF-1 on different solid culture media.

[0033] Figure 2 These are the SEM characteristics of the biocontrol fungus WZSF-1.

[0034] Figure 3 Antagonistic assay of WZSF-1 against E16 cells

[0035] Figure 4 The control effects of different treatments on pepper root-knot nematodes; A: root knot index; B: control effect;

[0036] Figure 5Figure 3 Effects of different treatments on pepper growth indicators, A: chlorophyll content; B: plant height; C: stem diameter; D: fresh weight of aboveground parts; E: root weight; "*" indicates significant difference at the 0.05 level; "**" indicates significant difference at the 0.01 level; "***" indicates significant difference at the 0.001 level.

[0037] Figure 6 Shows the growth of the aboveground parts of peppers with different treatments.

[0038] Figure 7 The effects of different treatments on pepper roots.

[0039] Figure 8 Effects of different treatments on PAL activity in pepper leaves.

[0040] Figure 9 Effects of different treatments on CAT enzyme activity in pepper leaves.

[0041] Figure 10 The effects of different treatments on PPO activity in pepper leaves.

[0042] Figure 11 Effects of different treatments on MDA content in pepper leaves

[0043] Note: Figure 4-11 In the table, CK is the blank control; AW is a 1000-fold dilution of 5% avermectin; E1 is the fermentation supernatant of Paecilomyces lilacinus E16; F1 is the fermentation supernatant of Streptomyces WZSF-1; H1 represents the combined fermentation supernatant of Paecilomyces lilacinus E16 and Streptomyces WZSF-1; E2 represents a 5-fold dilution of the fermentation supernatant of Paecilomyces lilacinus E16; F2 is a 5-fold dilution of the fermentation supernatant of Streptomyces WZSF-1; H1 represents a 5-fold dilution of the combined fermentation supernatant of Paecilomyces lilacinus E16 and Streptomyces WZSF-1.

[0044] Biomaterial Deposit

[0045] Deposit number: GDMCC No.65368

[0046] Name: Streptomyces griseorubens WZSF-1

[0047] Taxonomic nomenclature: Streptomyces griseorubens

[0048] Depository: Guangdong Provincial Microbiological Culture Collection Center (GDMCC)

[0049] Address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou

[0050] Storage time: October 30, 2024

[0051] Survival: Yes. DETAILED DESCRIPTION

[0052] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods. The materials and reagents used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0053] Test nematodes: The tested root-knot nematodes were isolated by the Vegetable Disease Research Laboratory, Institute of Environment and Plant Protection, Chinese Academy of Tropical Agricultural Sciences, identified according to conventional methods, and propagated and preserved in pepper roots.

[0054] Test culture medium

[0055] The test media included LB, PDA, fermentation basal medium, Gao's No. 1, and YE medium. The formulas of each medium are shown in Table 1.

[0056] Table 1. Basic culture medium and formula

[0057]

[0058] Example 1. Acquisition and identification of Streptomyces griseorubens WZSF-1

[0059] 1. Screening of Actinomycetes for Root-Knot Nematode Biocontrol

[0060] 1. Obtaining the second-instar larvae of the root-knot nematode

[0061] The roots of peppers that have been colonized by root-knot nematodes are pulled out, the surface soil is washed with clean water, and the root knots are cut into small pieces with scissors; the cut root knots are immersed in clean water, and clean water and sodium hypochlorite solution are added in a volume ratio of 200:1.5, and stirred with a meat grinder for 2-4 times, and then collected in a container and shaken for 5-8 minutes to soften the roots; 100-mesh, 300-mesh, and 500-mesh sieves are stacked from top to bottom, and the softened roots are poured into them. The roots are rinsed with clean water and rubbed by hand to break the root-knot nematode eggs; the 500-mesh sieve is repeatedly rinsed with running water to remove residual sodium hypochlorite until there is no irritating smell, and the root-knot nematode eggs on the 500-mesh sieve are collected and placed on a double layer of lens paper, immersed in clean water and incubated at room temperature. The hatching status can be observed daily under a dissecting microscope; finally, the hatched second-instar larvae are collected and prepared into an adult suspension for later use.

[0062] 2. Preparation of strain fermentation broth

[0063] 1) Seed solution preparation

[0064] 100 μL of actinomycetes stored in glycerol at -80°C was spread on Gao's No. 1 plate and cultured at 28°C for 7-15 days. A single colony was picked and cultured in 100 mL of YE medium at 28°C for 3 days with shaking for later use.

[0065] 2) Fermentation broth preparation

[0066] To prepare actinomycete fermentation broth, inoculate 1% seed solution of each strain into Gao's No. 1 liquid medium and culture at 28°C with shaking for 7 days. Centrifuge the fermentation broth at 10,000 rpm for 10 minutes to obtain the fermentation supernatant, which was stored at 4°C until needed.

[0067] 3. Primary screening and rescreening of nematicidal biocontrol bacteria

[0068] The activity of biocontrol bacteria against J2 was determined by the immersion method. 900 μL of the supernatant of the actinomycete fermentation solution was added to a 24-well plate, and then 100 μL of a suspension containing approximately 100 J2 was added and mixed. The mixture was then treated at room temperature for 24 hours. A blank medium was used as a control, and three replicates were set for each treatment. After treatment, the treated solution was collected into a centrifuge tube and diluted to 8 mL with clean water. The tube was centrifuged at 2000 rpm for 2 minutes, the upper layer of solution was removed, and the volume was diluted to 8 mL with clean water and resuspended for 12 hours. The number of nematode deaths was counted under a dissecting microscope. Nematodes that were immobile and unresponsive to mechanical touch were considered dead. The mortality rate was calculated according to the following formula:

[0069] Nematode mortality rate = number of dead nematodes / total number of tested nematodes × 100%

[0070] Corrected mortality rate = (treatment nematode mortality rate - control nematode mortality rate) / (1 - control nematode mortality rate) × 100%

[0071] Strains with high nematicidal activity from the initial screening results were re-fermented in shake flasks to determine their nematicidal activity. The nematicidal activity of the strains was measured after 3, 6, and 48 hours of treatment. The fermentation supernatant was diluted 2-fold and 5-fold to determine its activity. This allowed the identification of biocontrol strains with high and stable nematicidal activity.

[0072] The results, shown in Table 2, were obtained by the immersion method, revealing seven actinomycete strains with nematicidal activity against second-instar larvae of the root-knot nematode R. aurantii. Two strains, WZSZJF-2 (9.20) and WZSF-1, exhibited nematicidal activity exceeding 80% in their fermentation supernatant solutions over 24 hours. Their average corrected mortality rates against nematodes were 87.42 ± 1.30% and 88.08 ± 5.65%, respectively. Therefore, WZSZJF-2 (9.20) and WZSF-1 were selected for subsequent rescreening experiments.

[0073] Table 2. Results of the initial screening of nematicidal strains

[0074] strain number Average adjusted mortality rate (%) CK 0.00±0.00f WZSF-9 75.98±2.33ab WZSZJF-1 76.06±6.30ab WZSZJF-2(9.20) 87.42±1.30a WZSZJF-2(11.22) 77.05±9.37ab WZSF-1 88.08±5.65a WZSZJ-7 71.08±7.79b WZSZJ-5 79.29±1.56ab

[0075] Note: Different letters in the table indicate significant differences in nematicidal activity among different strains (P<0.05)

[0076] Two strains with nematicidal activity greater than 80% in the initial screening results were re-fermented in shake flasks, and their fermentation supernatants were assayed for nematicidal activity at 3, 6, 24, and 48 hours. The fermentation supernatants were also diluted 2-fold and 5-fold to assay their 24-hour nematicidal activity. The results are shown in Tables 3 and 4. The average corrected mortality rates of both strains were greater than 70% when diluted 2-fold, with the average corrected mortality rate of WZSF-1 reaching 81.16±4.04%, and its nematicidal activity was significantly higher than that of the other strains. Even after a 5-fold dilution, the average corrected mortality rate of WZSF-1 remained above 70%, significantly higher than that of the other strains. The fermentation supernatant of each strain was treated with nematodes for 3, 6, 24, and 48 hours. The nematicidal activity of all four strains increased with treatment time. The average corrected mortality rate of WZSF-1 was significantly higher than that of the other strains. At 3 hours of treatment, the average corrected mortality rate of WZSF-1 against J2 reached 80.65±4.04%. After 6 hours of treatment, the nematicidal activity of WZSF-1 was not much different from that at 3 hours. After 24 and 48 hours of treatment, the nematicidal activity of WZSZJF-2 (9.20) and WZSF-1 exceeded 80%, but the difference was not significant. Based on the results of the initial and rescreening, the fermentation supernatant of WZSF-1, its dilutions, and different treatment times all showed good nematicidal activity. The average nematicidal activity of the initial and rescreened stock solutions was 85.94% at 24 hours, thus identifying the strain WZSF-1 with high nematicidal activity.

[0077] Table 3 Effects of different dilution ratios on nematicidal activity

[0078]

[0079] Note: Different lowercase letters in the table indicate significant differences in the nematicidal activity of different strains (P<0.05), the same below.

[0080] Table 4. Changes in nematocidal activity at different treatment times

[0081]

[0082] 2. Classification and Identification of Biocontrol Bacteria

[0083] 1. Morphological characterization and scanning electron microscopy observation

[0084] The culture medium and its formula required for identification of colony morphology and culture characteristics are shown in Table 5.

[0085] Table 5. Culture characteristics of the medium

[0086]

[0087]

[0088] Morphological Observation: Prepare ISP culture media and Gao's No. 1 inverted plates. Inoculate WZSF-1 onto ISP and Gao's No. 1 plates and invert at 28°C for 7-10 days. Observe the colonies of the biocontrol fungus WZSF-1 for color, shape, size, and the presence of soluble pigment according to the "Rapid Identification and Systematic Classification of Actinomycetes" and "Identification Methods for Streptomyces" (Shirlin and Gottlieb 1966).

[0089] Scanning electron microscopy observation: The scanning electron microscopy observation of WZSF-1 was carried out using the insert method (Zhao Bin et al. 2014). After WZSF-1 was streaked on Gao's No. 1 plate, a sterilized cover slip was inserted into the culture medium at a 45° angle perpendicular to the streaking direction and inverted and cultured at 28°C for 5-7 days. The cover slip was then gently pulled out and air-dried. The mycelial growth part was cut into squares of approximately 0.5 cm × 0.5 cm in size. After the sample was dried, it was gold-plated and the mycelium, conidiophores and spore morphology of the strain were observed using a scanning electron microscope.

[0090] The results are shown in Table 6 and Figure 1 As shown, the biocontrol fungus WZSF-1 grew on ISP2, ISP3, ISP4, ISP5, Gao's No. 1, and PDA media, but was unable to grow on Czapek's agar and produced no soluble pigment on any of the media. Aerial hyphae were white on ISP3 and Gao's No. 1, while basal hyphae were pale yellow and white, respectively. On ISP2, both aerial and basal hyphae were yellow. On ISP4, aerial hyphae were off-white, while basal hyphae were white. On ISP5 and PDA, both aerial and basal hyphae were yellow.

[0091] Table 6 Culture characteristics of strain WZSF-1 on different solid media

[0092]

[0093]

[0094] Note: “+++” indicates good growth; “++” indicates average growth; “+” indicates weak growth; “-” indicates no growth.

[0095] The mycelial and spore morphology of WZSF-1 were observed by SEM ( Figure 2 The aerial hyphae of actinomycete WZSF-1 are long, flat, compact, and intricately interwoven. The tips of the aerial hyphae differentiate into spiral conidia, which produce many spores in the form of short rods with thorn-like protrusions on the surface.

[0096] In summary, based on the morphological and cultural characteristics, mycelial morphology, and physiological and biochemical characteristics of the strain, it was identified as Streptomyces griseorubens.

[0097] The above-mentioned strain WZSF-1 was deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou) on October 30, 2024, with the deposit number: GDMCC No. 65368, name: streptomyces griseorubens WZSF-1; classification name: streptomyces griseorubens.

[0098] Example 2. Test Materials and Methods

[0099] 2.1 Test materials

[0100] 2.1.1 Test strains and nematode sources

[0101] Test strains: The test actinomycetes WZSF-1 and Paecilomyces lilacinus E16 strains were isolated, identified, and preserved by the Microbial Resources and Utilization Laboratory, Institute of Environment and Plant Protection, Chinese Academy of Tropical Agricultural Sciences. The deposit number of Paecilomyces lilacinus E16 in the General Microbiology Center of the China Culture Collection Administration is CGMCC No. 5951; it is recorded in patent document CN102851219A.

[0102] Nematodes: The tested root-knot nematodes, R. incognita and R. incognita, were isolated and propagated in pepper roots by the Vegetable Disease Research Laboratory, Institute of Environment and Plant Protection, Chinese Academy of Tropical Agricultural Sciences, and then identified.

[0103] Pepper seedlings: Pepper seeds purchased from the market were cultivated in the laboratory as experimental materials.

[0104] 2.1.2 Test culture medium

[0105] The test media included LB, PDA, fermentation basal medium, Gao's No. 1, and YE medium. The formulas of each medium are shown in Table 1.

[0106] 2.1.3 Main reagents

[0107] Phenylalanine ammonia lyase kit (Solebol), catalase kit (Solebol), malondialdehyde kit (Solebol).

[0108] 2.2 Nematicidal activity assay

[0109] 2.2.1 Obtaining the second-instar larvae of the root-knot nematode

[0110] The roots of peppers that have been colonized by root-knot nematodes are pulled out, the surface soil is washed with clean water, and the root knots are cut into small pieces with scissors; the cut root knots are immersed in clean water, and clean water and sodium hypochlorite solution are added in a volume ratio of 200:1.5, and stirred with a meat grinder for 2-4 times, and then collected in a container and shaken for 5-8 minutes to soften the roots; 100-mesh, 300-mesh, and 500-mesh sieves are stacked from top to bottom, and the softened roots are poured into them. The roots are rinsed with clean water and rubbed by hand to break the root-knot nematode eggs; the 500-mesh sieve is repeatedly rinsed with running water to remove residual sodium hypochlorite until there is no irritating smell, and the root-knot nematode eggs on the 500-mesh sieve are collected and placed on a double layer of lens paper, immersed in clean water and incubated at room temperature. The hatching status can be observed daily under a dissecting microscope; finally, the hatched second-instar larvae are collected and prepared into an adult suspension for later use.

[0111] 2.2.2 Preparation of strain fermentation broth

[0112] 1) Strain activation

[0113] The preserved Paecilomyces lilacinus E16 was inoculated on PDA plates, and Streptomyces WZSF-1 was inoculated on Gao's No. 1 plates, and cultured at 28°C for 5 to 7 days.

[0114] 2) Seed solution preparation

[0115] a. Preparation of Actinomycete Seed Solution

[0116] A single colony was picked from the activated actinomycete plate with a sterile toothpick and cultured in 100 mL YE medium at 28°C with shaking for 3 days.

[0117] b. Preparation of fungal seed solution

[0118] Pick a single fungal colony and culture it in 100 mL of PDB medium at 28°C with shaking for 3 days.

[0119] 2.2.3 Fermentation broth preparation

[0120] The seed solution of each strain was inoculated into PDA and Gao's No. 1 liquid medium at a volume ratio of 1%, and cultured at 28°C with shaking for 7 days. The fermentation broth of each strain was centrifuged at 10,000 rpm for 10 minutes to obtain the fermentation supernatant of the strain, which was stored at 4°C for later use.

[0121] 3. Determination of Nematicidal Activity

[0122] The activity of the biocontrol bacteria against the second-instar larvae of the ear bean root-knot nematode was determined using the immersion method. 900uL of the fermentation supernatant or dilution of the strain was added to a 24-well plate, and then 100uL of a suspension containing approximately 100 second-instar larvae of the elephant ear bean root-knot nematode was added and mixed. The mixture was then incubated at room temperature for 24 hours. A blank medium was used as a control, and three replicates were set for each treatment. After treatment, the treated solution was collected into a centrifuge tube and diluted to 8mL with clean water. The tube was centrifuged at 2000r / min for 2 minutes, the upper layer was removed, and the solution was diluted to 8mL with clean water and resuspended for 12 hours. The number of nematode deaths was counted under a dissecting microscope. Nematodes that were immobile and unresponsive to mechanical touch were considered dead. The mortality rate was calculated according to the following formula:

[0123] Nematode mortality rate = number of dead nematodes / total number of tested nematodes × 100%

[0124] Corrected mortality rate = (treatment nematode mortality rate - control nematode mortality rate) / (1 - control nematode mortality rate) × 100%

[0125] 2.3 Study on the combined application of biocontrol bacteria and Paecilomyces lilacinus

[0126] 2.3.1 Flat plate standoff test

[0127] Referring to the method of (Wang Bo 2006), single colonies of Paecilomyces lilacinus E16 and actinomycete WZSF-1 were picked and inoculated into PDB medium and Gao's liquid medium No. 1 respectively. After culture in a constant temperature shaking incubator at 28℃ for 5-7 days, 10uL of E16 bacterial liquid was aspirated in the center of the PDA plate, and 10uL of actinomycete liquid was aspirated and placed symmetrically around it. Plate 1 without actinomycete liquid around it was used as a control. Five replicates were set for each treatment, and the inhibition rate was determined after 7 days of culture.

[0128] 2.3.2 Evaluation of nematicidal activity of compound with Paecilomyces lilacinus

[0129] The fermentation supernatant of Paecilomyces lilacinus E16 and the nematicidal biocontrol bacteria were mixed evenly in different volume ratios of 4:1, 2:1, 1:1, 2:1, and 4:1, and the nematicidal activity was determined according to "2.2.3 Determination of Nematicidal Activity". At the same time, the fermentation supernatant mixtures of different ratios were diluted to different multiples of 5 times and 10 times, and the nematicidal activity was determined according to "2.2.3 Determination of Nematicidal Activity".

[0130] 2.4 Potted plant prevention efficacy test

[0131] 2.4.1 Potted experiment design

[0132] Pepper seedlings of uniform size at the 3-4 leaf stage were transplanted into pots 8 cm high and 9 cm in diameter, and treated after one week of acclimatization. This experiment set up 9 treatments in total, namely: 1) Blank group (KB): no nematodes, only water treatment; 2) Control group (CK): 2000 nematodes were inoculated per pot and sterile fermentation medium was applied; 3) Treatment 1 (AW): 2000 nematodes were inoculated and a 1000-fold dilution of 5% avermectin emulsifiable concentrate was applied; 4) Treatment 2 (E1): 2000 nematodes were inoculated and the fermentation supernatant of Paecilomyces lilacinus E16 was applied; 5) Treatment 3 (F1): 2000 nematodes were inoculated and the fermentation supernatant of biocontrol bacteria was applied ; 6) Treatment 4 (H1): inoculation of 2000 nematodes and application of the composite fermentation supernatant of E16 and biocontrol bacteria, 7) Treatment 5 (E2): inoculation of 2000 nematodes and application of E16 plus a 5-fold dilution of the supernatant; 8) Treatment 6 (F2): inoculation of 2000 nematodes and application of a 5-fold dilution of the fermentation supernatant of biocontrol bacteria; 9) Treatment 7 (H2): inoculation of 2000 nematodes and application of a 5-fold dilution of the composite solution of E16 and biocontrol bacteria fermentation supernatant. 100 mL was applied to the roots of each pot for each treatment, and each treatment was repeated for 6 pepper seedlings.

[0133] 2.4.2 Statistics on prevention and control effects

[0134] 2.4.2.1 Determination of protective efficacy

[0135] On the 60th day after treatment, the pepper seedlings were pulled out, the soil around the roots was washed clean with running water, and photos were taken. The root knot index of peppers was graded according to the following grading scale (Dube and Smart 1987) and the control effect was calculated.

[0136] Level 0: No Root Knot

[0137] Level 1: There are a small number of root knots, accounting for 1%-25% of the whole root system;

[0138] Level 2: The number of root nodes is moderate, accounting for 26%-50% of the total root system;

[0139] Level 3: The number of root nodes is large, accounting for 51%-75% of the whole root system;

[0140] Level 4: There are a large number of root nodes, accounting for 76%-100% of the entire root system.

[0141] Root knot index = ∑[(level × number of diseased plants in this level)] / (total number of plants surveyed × highest level) × 100%

[0142] Control effect = (root knot index of control group - root knot index of treatment group) / root knot index of control group × 100%

[0143] 2.4.2.2 Physiological index measurement

[0144] The growth indices of pepper were determined; chlorophyll content, plant height, stem diameter, aboveground fresh weight and root weight of pepper were measured 60 days after treatment.

[0145] 2.4.2.3 Determination of pepper defense enzyme activity

[0146] Extraction of crude enzyme solution: Chop the treated leaves and mix them evenly. Weigh 0.1g of leaves and place them in a mortar. Add 2mL of the extract in the kit. Grind thoroughly and transfer to a 1mL centrifuge tube. Centrifuge at 10,000g / min for 10min. Take the supernatant for use and store in a 4℃ refrigerator for short-term storage.

[0147] The contents of phenylalanine ammonia lyase (PAL), catalase (CAT), polyphenol oxidase (PPO), and malondialdehyde (MDA) in pepper leaves were determined according to the reagent instructions.

[0148] 2.5 Data Analysis Software

[0149] This experiment used the analysis software IBM SPSS Statistics 27, the variance analysis was performed using Duncan analysis, the data were sorted using WPS software, and the graphs were drawn using Graphpad prism 8.0.

[0150] 3 Results and Analysis

[0151] 3.1 Evaluation of the nematicidal effect of the combination of actinomycetes and Paecilomyces lilacinus

[0152] 3.1.1 Plate antagonism experiment

[0153] The antagonism between E16 and WZSF-1 was determined by plate antagonism. Figure 3 As shown, the antagonism between the two strains was low, and the antagonism rate of WZSF-1 against E16 was 5.49%.

[0154] 3.1.2 Evaluation of nematicidal activity at different ratios of actinomycetes and Paecilomyces lilacinus

[0155] The fermentation supernatants of the two strains were compounded at volume ratios of E16:WZSF-1 of 1:1, 1:2, 1:4, 2:1, and 4:1, and their nematicidal activities were measured. The results are shown in Table 7. By measuring the nematicidal activity of each ratio, it was found that the nematicidal activity of the 1:4 ratio was the highest, and the nematicidal activity of the stock solution was increased by 8.28% and 1.57% compared with E16 and WZSF-1; after dilution of the fermentation liquid by 2 times and 5 times, the nematicidal activity of the fermentation liquid at the 1:4 ratio was increased by 14.98%, 3.25%, 17.67%, and 0.96% compared with E16 and WZSF-1.

[0156] Table 7. Nematicidal activity assay of WZSF-1 and E16 complexes

[0157]

[0158]

[0159] Note: Different lowercase letters before “ / ” indicate significant differences in nematicidal activity among different dilutions of the same fermentation broth (P<0.05); Different lowercase letters after “ / ” indicate significant differences in nematicidal activity among different fermentation broths at the same dilution (P<0.05)

[0160] 3.2 Potted plant prevention effect experiment

[0161] 3.2.1 Effect of biocontrol bacteria combination on the control of root-knot nematodes of ear bean and root-knot nematodes of southern root-knot nematodes

[0162] The root knot index of peppers after two months of treatment is as follows Figure 5 As shown in the figure, the root knot indexes of CK, avermectin, E16 stock solution (fermentation supernatant), WZSF-1 stock solution (fermentation supernatant), composite stock solution (a mixture of the fermentation supernatant of Streptomyces WZSF-1 and the fermentation supernatant of Paecilomyces lilacinus E16 in a volume ratio of 4:1), 5-fold dilution of E16, 5-fold dilution of WZSF-1 and 5-fold dilution of composite solution were 100%, 18.75%, 56.25%, 50.00%, 18.75%, 56.25%, 56.25% and 43.75%, respectively, and the control efficacy on pepper was 81.25%, 43.75%, 50.00%, 81.25%, 43.75%, 43.75% and 56.25%, respectively. The control effect of the compound solution treatment was the same as that of avermectin. Compared with the use of E16 and WZSF-1 solution alone, the control effect of the compound solution increased by 85.71% and 62.50%, respectively. After dilution, the control effect of each strain decreased, but the control effect of the compound solution was still higher than that of the single treatment, which was 28.57% higher than that of E16 and WZSF-1.

[0163] 3.2.2 Impact on pepper growth

[0164] The results of each treatment on the growth of pepper are as follows Figure 6 As shown. The chlorophyll content, plant height, stem diameter, aboveground fresh weight, and root weight of AW, E16, WZSF-1, and the composite treatment group all had a promoting effect. After applying the fermentation supernatant of the biocontrol bacteria, the chlorophyll content was significantly increased compared with CK, and the composite treatment content was higher than that of the two strains alone. The chlorophyll content of the original solution and the 5-fold diluted solution were 53.478 and 55.57SPAD, respectively. Compared with the application of E16 and WZSF-1 original solutions alone, the composite treatment increased by 10.38% and 7.83%;

[0165] Plant height of peppers increased significantly after treatment with the combined solution. Treatment with WZSF-1 also significantly increased plant height, though the effect was lower than that of the combined treatment, although the difference was not significant. Application of the biocontrol agent increased pepper stem diameter compared to the control (CK) treatment, with E16 showing a significant increase, reaching 6.61 mm, and the combined treatment showing an extremely significant effect, reaching 7.10 mm. AW, E16, WZSF-1, and the combined treatment all significantly increased the aboveground fresh weight and root weight of peppers, promoting plant growth. The combined treatment showed a greater effect than E16 and significantly higher than the CK and WZSF-1 treatments.

[0166] 3.2.3 Effects on defense enzyme activity and malondialdehyde content

[0167] 3.2.3.1 Phenylalanine ammonia lyase (PAL) activity

[0168] from Figure 8 It can be seen that after the application of biocontrol bacteria fermentation, the PAL enzyme activity of pepper leaves treated with avermectin, E16, and WZSF-1 decreased. The PAL enzyme activity of pepper leaves treated with the composite stock solution increased compared with the CK, with the highest enzyme activity at 22.42 U / g, an increase of 17.31% compared with the CK, and the effect was significantly better than the drug treatment. After the dilution treatment, the enzyme activity content of each treatment was lower than that of the stock solution.

[0169] 3.2.3.2 Catalase (CAT) activity

[0170] The results of the effects of each treatment on the enzyme activity of pepper leaves are as follows Figure 9 After applying the fermentation supernatant of the biocontrol bacteria, the activity of the pepper's defense enzymes increased, and the activity of the defense enzymes increased with the increase in dilution multiple. The enzyme activities of the composite stock solution, E16, WZSF-1, and the 5-fold dilution of the composite solution were significantly higher than those of the CK treatment. After applying the stock solution, the enzyme activity of the composite treatment increased by 39.49% and 12.74% compared with the single application of E16 and WZSF-1. After 5-fold dilution, the enzyme activity of each strain treatment increased compared with the stock solution. After dilution, the enzyme activity of the F2 treatment was the highest, reaching 1072.85 U / g. The enzyme activity of the composite treatment was still significantly higher than that of the single E16 treatment.

[0171] 3.2.3.3 Polyphenol oxidase (PPO) activity

[0172] The results of PPO enzyme activity determination in pepper leaves are as follows Figure 10 After applying the original solution of each strain, the enzyme activity increased compared with CK. The enzyme activity of the compound treatment increased significantly, reaching 213.00 U / g, which was 73.45% and 77.50% higher than that of the E1 and F1 treatments, respectively, and the effect was extremely significant. After 5-fold dilution, the enzyme activity of the E16 and WZSF-1 treatments increased, significantly higher than that of the CK treatment. The enzyme activity of the compound treatment decreased compared with the original solution, but was still significantly higher than that of the CK and WZSF-1 treatments.

[0173] 3.2.3.4 Malondialdehyde (MDA) content

[0174] The results of MDA content determination in pepper leaves are as follows Figure 11 The MDA content in pepper leaves increased after application of AW and E16 fermentation supernatant solutions, compound solutions and their 5-fold dilutions, while the MDA content decreased after application of WZSF-1 solution, E16 5-fold dilutions and compound solutions, but the differences were not significant compared with CK.

[0175] 4 Discussions

[0176] Previous studies have shown that the fermentation supernatant of strain WZSF-1 exhibits excellent in vitro nematode activity against second-instar larvae of root-knot nematodes. To further explore its potential application in combination with E16, the two strains were evaluated for antagonism using plate-to-plate and titer assays. The inhibition rate of the actinomycete against E16 was 5.49%, indicating weak antagonism between the two strains. In vitro activity against J2 was determined by combining the actinomycete with the fermentation supernatant of Paecilomyces lilacinus E16 at varying ratios. When the volume ratio of E16 to WZSF-1 was 1:4, the stock solution exhibited nematicidal activity that was 8.28% higher than that of E16 and 1.57% higher than that of WZSF-1. Furthermore, after dilution of the solution by 2-fold and 5-fold, the nematicidal activity of the fermentation solution at the 1:4 ratio was increased by 14.98%, 3.25%, 17.67%, and 0.96% compared to that of E16 and WZSF-1, respectively.

[0177] To further explore the potential of the two strains and their combination against second-instar larvae, this study conducted pot experiments to verify their efficacy. Potted plant results showed that treatment with the biocontrol bacteria reduced the root knotting index of pepper roots. Compared with the CK treatment, the root knotting indexes of E16, WZSF-1, and the combination treatments were 56.25%, 50.00%, and 18.75%, respectively, representing control efficacies of 43.75%, 50.00%, and 81.25%, respectively. Compared with the E16 and WZSF-1 solutions alone, the combination solution increased its efficacy by 85.71% and 62.50%, respectively. While the efficacy of each strain decreased after dilution, the combination remained superior to the individual treatments, achieving a 28.57% increase compared to E16 and WZSF-1. Treatment increased the stem diameter, plant height, chlorophyll content, and aboveground and belowground fresh weight of peppers, demonstrating that the two strains exhibited effective control efficacy in potted peppers and promoted plant growth to a certain extent. There has been extensive research on the use of biocontrol bacteria to control root-knot nematodes, but the effectiveness of these applications has often been suboptimal. This may be due to the strong environmental tolerance and poor adaptability of individual strains. Studies have shown that combining biocontrol bacteria can improve their adaptability to the environment, while also enhancing the range and stability of their control effectiveness.

[0178] After applying biocontrol bacteria, the activity of pepper defense enzymes was measured. The results showed that the PAL, CAT, and PPO defense enzyme activities in the treated pepper leaves were higher than those in the CK, and the PAL activity of the combined treatment was significantly different from that in the CK. Treatment with each strain also increased the activity of pepper defense enzymes, but root irrigation with the original solution increased the MDA content of the pepper, which decreased after a five-fold dilution. After nematodes infect crops, the MDA content in the crops changes, reactive oxygen free radicals accumulate, and antioxidant capacity is suppressed, which changes the activity of defense enzymes and the degree of membrane lipid peroxidation in the leaves. Applying biocontrol bacteria can, to a certain extent, enhance the activity of defense enzymes in crops, improve their ability to resist disease, and play a protective role (Wang Binglin et al. 2012, Ye Deyou et al. 2012, Guo Jinghua 2007). 5 Conclusion

[0179] Root-knot nematodes are increasingly damaging crops. This study used second-instar larvae of root-knot nematodes as research subjects to screen strains with efficient and stable nematicidal activity. The effects of a combination of a nematicidal biocontrol bacterium and Paecilomyces lilacinus E16 on the nematicidal activity were also evaluated. Pot experiments were conducted to evaluate the control efficacy of the biocontrol bacterium, E16, and the composite fermentation liquid against root-knot nematodes.

[0180] (1) WZSF-1 was mixed with Paecilomyces lilacinus E16 in different proportions and their nematicidal activity was measured. It was found that when the volume ratio of E16 to WZSF-1 was 1:4, the mixed solution had the highest nematicidal activity, and the nematicidal activity of the mixed solution was increased by 8.28% and 1.57% compared with E16 and WZSF-1 respectively. After being diluted 2 times and 5 times, the nematicidal activity of the fermentation liquid with a 1:4 ratio was increased by 14.98%, 3.25%, 17.67% and 0.96% compared with E16 and WZSF-1 respectively.

[0181] (2) The results of the potted plant experiment showed that WZSF-1, Paecilomyces lilacinus, and the combined solution all had good biocontrol effects on pepper root-knot nematodes, with the highest control effects reaching 43.75%, 50.00%, and 81.25%, respectively. Compared with the use of E16 and WZSF-1 alone, the combined treatment increased the control effect by 85.71% and 62.50%. After application, the chlorophyll content, stem diameter, and plant height of peppers were higher than those of the control treatment. The activities of defense enzymes were also higher after treatment than those of the control treatment.

Claims

1. A combined bacterial agent for controlling nematodes, the active ingredients of which are composed of the following components: 1) Streptomyces WZSF-1 or its fermentation supernatant, 2) Paecilomyces lilacinus E16 or its fermentation supernatant, The Streptomyces WZSF-1 is named as Streptomyces griseorubens WZSF-1, classified as Streptomyces griseorubens, and its preservation number in Guangdong Provincial Microbiological Culture Collection Center is GDMCC No. 65368.

2. The combined bacterial agent according to claim 1, wherein the volume ratio of the fermentation supernatant of Streptomyces WZSF-1 to the fermentation supernatant of Paecilomyces lilacinus E16 is 4:

1.

3. The combined bacterial agent according to claim 2, characterized in that: The preparation method of the Streptomyces WZSF-1 fermentation supernatant is as follows: 1) Preparation of Streptomyces WZSF-1 seed solution A single colony was picked from the activated Streptomyces WZSF-1 plate with a sterile toothpick and cultured in 100 mL YE medium at 28°C with shaking for 3 days to obtain the Streptomyces WZSF-1 seed solution; 2) Preparation of fermentation supernatant The seed liquid of Streptomyces WZSF-1 was inoculated into Gao's No. 1 liquid culture medium at a volume percentage of 1%, and cultured at 28°C for 7 days. The fermentation liquid of each strain was centrifuged at 10,000 r / min for 10 minutes to obtain the fermentation supernatant of Streptomyces WZSF-1.

4. The combined bacterial agent according to claim 2, characterized in that: The preparation method of the fermentation supernatant of Paecilomyces lilacinus E16 is as follows: 1) Preparation of Paecilomyces lilacinus E16 seed solution A single colony of Paecilomyces lilacinus E16 was picked and cultured in 100 mL of PDB medium at 28°C for 3 days with shaking to obtain the seed solution of Paecilomyces lilacinus E16; 2) Preparation of fermentation supernatant The seed liquid of Paecilomyces lilacinus E16 was inoculated into PDA liquid culture medium at a volume percentage of 1%, and cultured with shaking at 28° C. for 7 days. The fermentation liquid of each strain was centrifuged at 10,000 rpm for 10 minutes to obtain the fermentation supernatant of Paecilomyces lilacinus E16.

5. Use of the combined bacterial agent according to any one of claims 1 to 4 in controlling root-knot nematodes.

6. The use according to claim 5, characterized in that The root-knot nematodes are root-knot nematodes of the elephant ear bean family and root-knot nematodes of the southern region.

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