CN104894011A - Composite microbial agent for prevention and treatment of tomato root knot nematode disease and application thereof - Google Patents

Abstract

The invention relates to the technical field of crop disease prevention and treatment, in particular to a compound microbial agent for preventing and treating tomato root-knot nematode and its application. PGPR (Bacillus subtilis) is shared, utilizes the synergistic effect of both, compares with the control efficiency of general single bacterial agent or mycocin, the control rate of the present invention reaches 60-70%, is higher than single bacterial agent or mycocin Control rate. Simultaneously, the root system of the tomato plant applied with the compound bacterial agent is developed, especially the fibrous root system is more, which effectively increases the absorption area of the tomato.

Description

Compound microbial agent for controlling tomato root-knot nematode and its application

technical field

The invention relates to the technical field of crop disease prevention and treatment, in particular to a compound microbial bacterial agent for preventing and treating tomato root-knot nematode and its application.

Background technique

Root-knot nematode disease is a kind of worldwide important plant nematode disease caused by root-knot nematode (Meloidogyne spp.), and it is one of the important diseases of vegetables (tomato, cucumber, pumpkin, green pepper, etc.) everywhere. At present, there are more than 90 kinds of root-knot nematodes reported in the world, which mainly parasitize on more than 3,000 hosts such as vegetables, food crops, cash crops, fruit trees and weeds. Plants in temperate, subtropical, and tropical regions are particularly damaged. According to statistics in 2000, the annual losses caused by nematode damage to food and fiber crops in the world are about 12.7%, and the losses caused to vegetables, peanuts, tobacco and some fruit trees exceed 20%. Nematodes have become an important pathogen of plants, and nematode diseases have become an important problem in agricultural and forestry production. With the increase of vegetable production area in protected areas, especially since the large-scale promotion of solar greenhouses, the multiple cropping index has increased, and the heavy cropping has caused the damage of root-knot nematodes to become more and more serious. Generally, the yield is reduced by about 10%, and the severe one is as high as 75%.

Currently, root-knot nematode control measures are mainly divided into chemical pesticide control, physical control and biological control. For the control of root-knot nematodes, chemical pesticide control is currently the most widely used agricultural measure, usually using halogenated hydrocarbon compounds, methyl isothiocyanate, organophosphorus and carbamate compounds, these compounds can kill Kill or paralyze root-knot nematodes, but its disadvantages are obvious, which can lead to increased drug resistance of nematodes, increased population density, polluted environment, killed natural enemies, pesticide residues, increased drug resistance of pathogens and insects, and threatened human health. In addition to chemical pesticide control methods, another widely used method is physical control, mainly including: heat treatment, seed elimination, high frequency treatment, radio frequency treatment, high temperature stuffy shed, water and fertilizer management, soil improvement, material isolation and other methods. High-temperature stuffy shed, heat treatment, seed elimination, high-frequency treatment, and radio-frequency treatment are mainly used to treat seeds, seedbeds and seedlings in greenhouses, which may require a long period of shed rest, disrupt production plans, and affect crop growth cycles. It is suitable for controlling nematodes in field. Its application range is limited, and its control effect is average, and some require special equipment, which increases production costs. Therefore, in addition to continuing to explore some effective methods of chemical and physical control of root-knot nematodes, more and more attention should be paid to the biological control of root-knot nematodes. Root-knot nematode biocontrol resources include Nematophagous fungi, bacteria, Actinomycetes, Virus, Rickettsia, Protozoa, water bear ( Tardigrade), Turbellarians, Mites, Collembola, Enchytraids, Predacious nematodes, etc. Among them, Paecillin lilacin, Puccinia pachytansporum, Abamectin, Bacillus, etc. are more common, and the control efficiency is about 40%-60%. Single, resulting in a single biocontrol mechanism, no synergistic effect between bacterial strains, and no further improvement in control efficiency.

Contents of the invention

Aiming at the deficiencies in the prior art, the present invention provides a compound microbial bacterial agent for preventing and treating tomato root-knot nematode and its application, in which tomato symbiotic mycorrhizal (AM) fungi and plant rhizosphere growth-promoting bacteria (PGPR) are combined Shared, using the synergistic effect of the two to improve the control efficiency of root-knot nematodes.

Technical scheme of the present invention is:

A composite microbial agent, the active ingredient of which is composed of Glomus versiforme (G.v) and Bacillus subtilis (BS).

Among them, Glomus terrestris G.v is a kind of AM fungus, which lives symbiotically with plants, and was purchased from Mycorrhizal Technology Research Institute of Qingdao Agricultural University.

Bacillus subtilis (BS) belongs to the genus Bacillus and is a plant growth-promoting rhizobacteria (PGPR).

Plant growth-promoting rhizobacteria (PGPR) refers to a kind of beneficial bacteria that can fix nitrogen, decompose phosphorus, produce plant hormones or secrete antibiotics, and promote plant growth. . A large number of experiments at home and abroad have shown that inoculating PGPR in the root circle of crops can adjust the balance of soil microbial flora, and at the same time form a biological barrier in the root circle of plants to prevent the colonization and invasion of pathogens and promote plant growth. Its biocontrol mechanism is summarized as follows: PGPR competes with pathogens for water, nutrients and colonization sites; secretes siderophagen, which competes with pathogens for iron ions; some microorganisms produce antibiotics; induces plants to produce systemic resistance. sex etc. PGPR is one of the ideal biocontrol factors that can develop in the rhizosphere, and has great application potential in the current and future biological control of plant soil-borne diseases.

AM fungi are a type of plant biotrophic obligate symbiotic bacteria that exist in various terrestrial ecological environments and establish mutualistic symbiotic relationships with most plants. 90% of the vascular plants on the earth can form AM. The beneficial effects of mycorrhizae are mainly reflected in: expanding the absorption area of plant roots; increasing the absorption and utilization of phosphorus and other mineral elements by plants, improving the nutritional status; changing the balance of plant endogenous hormones, thereby promoting plant germination and rooting and growth; improve plant disease resistance and stress resistance; promote crop growth, increase yield, and improve quality.

There are at least three relationships between AM fungi and PGPR: mutual promotion, mutual competition and mutual non-influence. The species and environmental conditions of AM fungi, PGPR and host plant determine the nature of these interactions. Moreover, the interaction between AM fungi and PGPR is highly specific. AM fungi and PGPR have a series of interactions in the rhizosphere that are beneficial to plant growth and health and soil fertility, especially the synergy between them has very important physiological and ecological effects on plants and soil.

The biocontrol bacterial agent for preventing and treating tomato root-knot nematode disease is prepared by using the above-mentioned compound microbial bacterial agent.

The application of the biocontrol bacterium product in the prevention and control of tomato root-knot nematode.

Adopt the method for preventing and treating tomato root-knot nematode disease with the above-mentioned biocontrol agent, at first, in tomato seedling stage, inoculate Glomus surface G.v in seedling soil; Bacillus subtilis BS.

On the basis of the above scheme, the Gv inoculation dosage is 1000-5000 IPU/strain; the BS inoculation dosage is 10 ⁹ cfu/ml of fermentation broth, 200-500ml/strain·time.

Preferably, the Gv inoculation dosage is 2000 IPU/strain; the BS inoculation dosage is 10 ⁹ cfu/ml of fermentation broth, 200ml/strain·time.

Wherein, the transplanting period is the period when the tomato develops to 3-4 leaves; the seedling period is the stage when the tomato develops to 4-5 leaves after planting; the flowering and fruit-setting period is the period when the tomato develops to one flower and one fruit.

The beneficial effects of the present invention are:

1. Higher control efficiency: Compared with the control efficiency of general single bacterial agent or mycocin, the interaction between AM fungi and endophytic bacteria has obvious synergistic effect, and the control rate reaches 60-70%. The mechanism of inhibiting root-knot nematode is higher than the control rate of single bacterial agent or mycocin. This is closely related to the synergistic effect and extensive mechanism of action of the two bacterial agents.

2. Promote the development of tomato root system and increase yield: the tomato plants with this compound bacterial agent have well-developed root system, especially more fibrous root system, which effectively increases the absorption area of tomato.

3. The control method of the present invention is environmentally safe, beneficial to human health, does not kill natural enemies, has no residues, is not easy to produce resistance to pests and bacteria, is beneficial to the development of plant roots, does not require special equipment, does not require a shed period, and reduces Chemical pesticides have the advantages of damage and inhibition of tomato root system. The cost of using mixed bacterial agents is relatively low, far lower than the commonly used soil fumigant methyl bromide, etc., and there is no need to use special heating and vaporization equipment like methyl bromide, which can save a lot of expenses. The two bacterial agents are applied to the soil and will not remain in the fruit, which is safe and effective. It can not only prevent and control root-knot nematode diseases, protect and promote tomato root growth, but also increase yield and reduce pesticide pollution.

The present invention obtains the inhibitory effect of joint inoculation AM fungus and PGPR to tomato root-knot nematode disease by pot test and field test, draws following conclusions:

Pot experiments showed that combined inoculation of AM fungal strain G.v and PGPR strain BS could effectively inhibit tomato root-knot nematode disease. Compared with the single inoculation of G.v and BS, the incidence of root-knot nematode, the disease index and the number of root knots per plant in the combined inoculation treatment group were far less than those in the single inoculation treatment group, and the control effect reached 74%. The number of second instar larvae, the number of females in the root, the total number of nematodes in the root, the number of oocysts and the egg content of a single oocyst were all much lower than those in the single inoculation treatment group. It can be seen that the combined inoculation of G.v and BS can effectively inhibit tomato root-knot nematode disease . Part of the inhibition mechanism is to inhibit the root-knot nematode disease by inhibiting the infection of the second-instar larvae of root-knot nematodes and increasing the activity of the tomato's own resistance enzymes. The infection rate of root-knot nematodes in the control group was higher than that of the combined inoculation treatment group at 28 days. 4.15 times; combined inoculation treatment group POD, SOD, CAT and other resistant enzyme activities will generally be higher than the non-inoculation group and single inoculation treatment group, various resistance enzyme activities and active substance content present certain regular changes, general root enzyme activity and The content of active substances changed first, and the leaves changed later. The peak time and variation law of different active substances are different.

Detailed ways

The specific embodiment of the present invention is as follows:

Example 1:

The invention relates to a composite microbial agent, the active ingredients of which are composed of Glomusversiforme (G.v) and Bacillus subtilis (BS).

A biocontrol bacterial agent for preventing and treating tomato root-knot nematode is prepared by using the composite microbial bacterial agent.

The method for the prevention and treatment of tomato root-knot nematode by biocontrol fungicides, at first, in the tomato seedling cultivation stage, inoculate Glomus Gv in the seedling cultivation soil, the inoculation dose is 2000IPU/strain; again, in the tomato 3-4 leaf transplanting stage , seedling stage, and flowering and fruit-setting stage, the Bacillus subtilis BS was inoculated three times, and the BS inoculation dose was 10 ⁹ cfu/ml in the fermentation broth, 200ml/strain. The inoculation method of Bacillus subtilis is to make 4 small holes in the tomato root circle, and use the root irrigation method to inoculate.

Example 2:

The invention relates to a composite microbial agent, the active ingredients of which are composed of Glomusversiforme (G.v) and Bacillus subtilis (BS).

The biocontrol bacterial agent for preventing and treating tomato root-knot nematode is prepared by using the composite microbial bacterial agent.

The method for the prevention and treatment of tomato root-knot nematode by biocontrol agent, at first, in the tomato seedling cultivation stage, inoculate Glomus Gv in the seedling cultivation soil, the inoculation dose is 1000IPU/strain; Again, in the tomato 3-4 leaf transplanting stage , seedling stage, and flowering and fruit-setting stage, the Bacillus subtilis BS was inoculated three times, and the BS inoculation dose was 10 ⁹ cfu/ml of fermentation broth, 500ml/strain. The inoculation method of Bacillus subtilis is to make 4 small holes in the tomato root circle, and use the root irrigation method to inoculate.

Example 3:

The invention relates to a composite microbial agent, the active ingredients of which are composed of Glomusversiforme (G.v) and Bacillus subtilis (BS).

A biocontrol bacterial agent for preventing and treating tomato root-knot nematode is prepared by using the composite microbial bacterial agent.

The method for the prevention and treatment of tomato root-knot nematode by biocontrol agent, at first, in the tomato seedling cultivation stage, inoculate Glomus Gv in the seedling cultivation soil, the inoculation dose is 5000IPU/strain; Again, in the tomato 3-4 leaf transplanting stage , seedling stage, and flowering and fruit-setting stage, the Bacillus subtilis BS was inoculated three times, and the BS inoculation dose was 10 ⁹ cfu/ml of fermentation broth, 300ml/strain. The inoculation method of Bacillus subtilis is to make 4 small holes in the tomato root circle, and use the root irrigation method to inoculate.

Experimental example:

(1) The inhibitory effect and mechanism of inoculation of G.v+BS on tomato root-knot nematode under potted conditions

1. Test method

(Glomus versiforme, G.v), Bacillus subtilis (Bacillus subtilis) BS, and root-knot nematode (M.incognita Chitwood, Mi). identified as root-knot nematode incognita.

The experiment was divided into 8 treatments: CK, Mi, Gv, G.v+Mi, BS, BS+Mi, G.v+BS and G.v+BS+Mi. Inoculate 5000 IPU of Gv mixed inoculum when sowing, inoculate BS fermentation liquid 10 ⁹ cfu/ml 10ml in transplanting, and inoculate 500 root-knot nematode second-instar larvae/plant in transplanting. In addition to inoculating the corresponding inoculum, each treatment received an equal amount of other sterilized inoculum to maintain the consistency of each treatment group.

The tested tomato seeds were sterilized by 2% NaClO and sown in black nutrient pots (the ratio of soil to sand was 2:1). When the tomato seedlings grew to the 3-leaf stage, they were transplanted into flowerpots. 2 plants per pot, 20 pots per treatment as a test plot, 5 plants were randomly selected from each plot to measure each index.

The G.v, BS and Mi inoculation methods, the preparation method of BS fermentation broth and the management method of tomato seedlings are as follows.

Inoculation of AM fungi: Put the cultivation soil (soil and sand ratio of 2:1) into the black nutrient bowl and mix with 5000 inoculum potential units (IPU) of AM fungal agent, and add the same amount of sterilized mixed inoculum as the control to maintain The same for other rhizosphere microflora. Two tomato seedlings (1 leaf stage) were planted in each pot. normal management.

Preparation of root-knot nematode egg suspension: Pick fresh egg masses from tomato roots infected with root-knot nematode, shake vigorously with 2% NaClO solution for 2 minutes, recover eggs through a 500-mesh sieve, rinse several times with sterile water, and dilute with sterile water into 600 eggs/ml.

Preparation of PGPR fermentation broth: transfer 10 μl of strains into a 250ml Erlenmeyer flask filled with 50ml of liquid medium, and culture with shaking at 30°C and 130rpm/min for 48h. Dilute to 10 ⁹ cfu/ml by turbidimetric method.

Inoculation of Mi and PGPR: Tomatoes were transplanted into pots with a diameter of 20 cm at the 3-4 leaf stage, with 2 plants per pot, repeated 6 times. At the same time, 4 small holes were made around the tomato root, and the root irrigation method (combination of surface and deep layers) was used to inoculate Mi 5ml and PGPR 10ml. According to the fertility level of the culture substrate and the needs of plant growth, 30% Hoagland nutrient solution can be properly supplemented in the middle and late stages. Other routine management.

2. Measuring indicators and methods

Tomato seedling root and leaf samples were randomly taken at 0, 2, 4, 7, 14, 21, and 28 days after transplanting, frozen in liquid nitrogen, and stored at -80°C for later use.

Root-knot nematode incidence: control effect (%) = (disease index of single inoculation with Mi - disease index of inoculation with G.v and BS) / disease index of single inoculation with Mi * 100%

Determination of the disease index: the disease condition adopts the grading standard of 0-6 grades. Grade 0, no root nodes are formed; Grade 0.5, the number of roots forming root knots accounts for less than 10% of the total number of roots, the root knots are small, and the root development is almost unaffected; Grade 1, the number of roots forming root knots accounts for 10% of the total number of roots % or less, the root knots are relatively large, and root clusters are formed; level 2, the number of roots forming root knots accounts for 10% to 20% of the total number of roots, the root knots are medium, and root clusters are more obvious; level 3, root formation The number of roots with knots accounts for 20% to 50% of the total number of roots, and some root knots converge to form obvious root expansion; Grade 4, the number of roots that form root knots accounts for 50% to 70% of the total number of roots, most of the root knots converge and form obvious root system expansion; grade 5, the number of roots forming root knots accounts for 70% to 90% of the total number of roots, there are only a few fibrous roots, and the rest are root knots converging to form obvious root expansion; grade 6, the number of roots forming root knots Accounting for 100% of the total root system, there are no fibrous root systems, and the root systems have obvious rot symptoms. The disease index, incidence rate and control effect were calculated by the following formulas:

Disease index (%) = ∑ (each disease level × number of plants at all levels) × 100% / (highest disease level × total number of investigated plants)

The number of nematodes in the root: Sodium hypochlorite-acid fuchsin staining, add an appropriate amount of 5.25% NaClO solution to the root tissue, stir continuously for 4 minutes, take it out, rinse with running water for 45 seconds, soak in distilled water for 15 minutes, then move the root tissue to another beaker, add 1ml Acid fuchsin solution, boiled for 30S, rinse with running water after cooling, and finally add 25ml of acid glycerin solution to the root tissue, fade for microscopic examination.

Number of oocysts: The root system of tomato was evenly cut into 1cm root segments, 1g was weighed randomly, and the number of oocysts was recorded.

The number of eggs in a single oocyst: about 10 oocysts were dissolved in 5% sodium hypochlorite for 3 minutes, and then the number of eggs in each oocyst was counted.

Determination of the number of root knots per plant: Take all the roots of tomatoes, and use a counter to count the number of root knots.

3. Test results

(1) Effects of G.v and BS on the incidence of tomato root-knot nematode

Table 1 Effect of G.v and BS on the incidence of tomato root-knot nematode

Table 1 shows that G.v and BS can effectively alleviate tomato root-knot nematode disease, the incidence rate decreased by 23% and 50%, the disease index decreased by 30.3% and 47%, and the control effect reached 37.76% and 58.51%. Under these conditions, BS had a better disease resistance effect; while the G.v+BS mixed treatment group showed a better synergistic advantage, and the disease incidence and disease index control effect were higher than those of the single inoculation treatment group.

(2) Effects of G.v and BS on propagules of root-knot nematode

G.v and BS can effectively alleviate tomato root-knot nematode disease, and the root-knot nematode propagule indicators such as the total number of nematodes in the root, the number of oocysts, and the egg content of a single oocyst also decreased (Table 2), which were significantly lower than those of the Mi treatment group. Compared with the single inoculation treatment group, the G.v+BS mixed treatment group can more effectively reduce the number of second instar larvae (22.2/g), the number of females (50.6/g) and the total number of nematodes (72.8) in roots. /g), the number of oocysts (20.0/g) and the egg content of a single oocyst (333.2), reduce the infection rate of second-instar larvae of root-knot nematode and reduce its effective reproduction rate.

Table 2 Effects of G.v and BS on propagules of M. incognita

Note: Different letters in the table indicate that different treatments are significantly different at p=0.01 level.

(3) Infection rate of M. incognita with different treatments

It can be seen from Table 3 that the root-knot nematode infestation rate of G.v, BS single inoculation treatment group and mixed inoculation group gradually decreased with the passage of time, and the decline was obvious at 14 days after transplanting, while the Mi treatment group was the opposite. The infection rate of root-knot nematodes increased rapidly at 14 days, and was close to the maximum infection rate. The results showed that tomato symbiotic bacteria G.v and endophytic bacteria BS had significant effects on the reproductive activity and infection process of root-knot nematodes about 14 days after transplanting. Obvious inhibitory effect; the root-knot nematode infection rate of G.v+BS mixed inoculation group was significantly lower than that of other treatment groups at each time period, and it was only 0.826 at 28 days, which was 4.16 times that of Mi treatment group, G.v treatment group and BS treatment group respectively , 1.49 times and 1.85 times.

Table 3 Different treatment M. incognita infestation rate (%day)

(4) Effect of G.v+BS inoculation on tomato resistant enzymes

Under adversity conditions, plants will resist adverse factors by improving their own resistance, increasing the activity of resistant enzymes, the content of active substances, and reducing the generation of harmful substances. The infection of root-knot nematodes to tomato roots will also cause corresponding reactions in tomato roots and leaves. It can be seen from the table below that the activities of resistant enzymes such as POD, SOD, and CAT in the treatment group treated with root-knot nematodes are generally higher than those in the non-inoculated group, while In the single-inoculation Mi treatment group, with the infection of root-knot nematodes, the activities of various resistant enzymes and the content of active substances showed certain regular changes. Generally, the enzyme activity and content of active substances in the root system changed first, and the leaves changed later. The peak time and variation law of different active substances are different.

1) Effect of G.v+BS inoculation on tomato root and leaf peroxidase (POD)

It can be seen from Table 4-5 that the POD activity of each treatment group showed a certain regularity with the increase of days after transplanting, and the Mi-inoculated treatment group was generally higher than the corresponding non-Mi-inoculated treatment group; There was a significant change in the 7th day, and then the POD activity of the CK treatment group increased, but the change curve was gentle. The BS treatment group was higher than the CK treatment group on the 7th day. It was significantly higher than that of CK and BS treatment groups at 4 days after planting, and reached the peak at 14 days, which were 66.29 (△OD ₄₇₀ ·min ^-1 ·g ^-1 ) and 57.25 respectively; The inoculated Mi treatment group was close, but the former was significantly higher than the latter, and the second peak appeared on the 28th day, which may be related to the secondary infection of root-knot nematode; the POD activity of tomato leaves was also on the 7th day There was a significant change, but the peak value was not obvious. Various infection factors had little effect on the POD activity of tomato leaves, and the Mi treatment group also increased significantly at 28 days.

Table 4 POD activity of tomato roots under different treatments (△OD ₄₇₀ ·min ^-1 ·g ^-1 )

Table 5 POD activity of tomato leaves under different treatments (△OD ₄₇₀ ·min ^-1 ·g ^-1 )

2) Effect of G.v+BS inoculation on tomato root and leaf catalase (CAT)

It can be seen from Table 6-7 that the CAT activity of tomato roots and leaves changed significantly with the increase of transplanting days. In the non-inoculated Mi treatment group, the CAT activity of the root system in the CK treatment group did not change significantly, showing a relatively gentle change curve, and decreased slightly after the 7th day, but the BS, Gv and G.v+BS mixed inoculation treatment groups could all be inoculated. Significantly increased root CAT activity, among which Gv and G.v+BS mixed inoculation treatment groups were close, and two peaks appeared on the 7th and 21st days, while BS 51-3 gradually decreased after the 7th day; the inoculation Mi treatment group generally It was higher than that of the non-vaccinated treatment group, and the trend of each group was close. There were also two peaks on the 7th day and the 21st day, and the Gv and G.v+BS mixed inoculation treatment groups were still higher than other treatment groups. On the 7th day, the The peak values were 3.868 and 4.063 (△OD ₂₄₀ ·min ^-1 ·g ^-1 ), and the peak values were 3.563 and 3.766 (△OD ₂₄₀ ·min ^-1 ·g ^-1 ) on the 21st day. The change trend of CAT activity in tomato leaves is different from that in roots. There is only one peak, which appears on the 7th day. On the 14th day, it indicated that the infection of Mi and the infection of Gv and BS interacted with each other, and the comprehensive infection result was very complicated.

Table 6 CAT activity of tomato roots under different treatments (△OD ₂₄₀ ·min ^-1 ·g- ¹ )

Table 7 CAT activity of tomato leaves with different treatments

3) Effect of G.v+BS inoculation on tomato root and leaf superoxide dismutase (SOD)

It can be seen from Table 8-9 that the infection of Mi, Gv and BS had certain effects on the SOD activity of tomato roots and leaves. The most obvious change in root SOD activity was 7 days after transplanting. In the non-inoculated Mi treatment group, the change of CK was not obvious. In the BS treatment group, the root SOD activity increased on the 4th day, then decreased on the 7th day, and then rose slowly. The difference is that the Gv and G.v+BS mixed inoculation treatment groups peaked at 7 days, respectively 977.86U·g ^-1 and 1035.14U·g ^-1 , which were 1.26 and 1.33 times that of the corresponding CK treatment group, and then slowly decline. The change trend of SOD activity in leaves was basically the same as that in roots, and the SOD activity in leaves was significantly higher than that in roots. The magnitude of change in the Mi-vaccinated treatment group was smaller than that in the non-vaccinated treatment group.

Table 8 SOD activity of tomato roots in different treatments (U·g ^-1 )

Table 9 SOD activity of tomato leaves under different treatments (U·g ^-1 )

(2) The inhibitory effect of G.v and BS inoculation dose and inoculation timing on tomato root-knot nematode

1. Effect of G.v inoculation dosage on AM fungal infection rate before planting tomato seedlings

(1) Experimental design

A total of 4 treatments were set up: control group (CK), treatment groups inoculated with G.v 500, 1000, 2000 and 5000 IPU respectively. Each treatment group was inoculated with G.v bacterial agent when sowing. Sow two sterilized and germinated tomato seeds in the seedling soil premixed with AM bacterial agent, 2 plants per pot, 6 pots per treatment, and normal management. When the tomato seedlings grow to the 3-4 leaf stage, samples are taken to determine the mycorrhizal infection rate.

(2) Determination method of mycorrhizal infection rate

Cut the root segment into 0.5-1.0 cm small segments, add 10% KOH solution to make it transparent, and put it in a 90°C water bath for 15-20min. Remove the lye, rinse the root system with tap water 3 times, and add 2% HCl solution to acidify for 5 minutes. After removing the acid solution, add acid fuchsin (0.1%) lactoglycerin staining solution (lactic acid 875ml, glycerin 63ml, distilled water 63ml, acid fuchsin 0.1g), overnight at room temperature. Microscopic examination can be performed after adding lactic acid for color separation.

Mycorrhizal infection rate (%)=∑(0%×number of root segments+10%×number of root segments+…+100%×number of root segments)×100%/total number of root segments

(3) Results

The mycorrhizal infection rate of table 10 inoculating different doses of G.v tomato seedlings

2. Inhibitory effect of BS inoculation dose, inoculation timing and inoculation frequency on tomato root-knot nematode disease

(1) Experimental design

Inhibitory effect of BS inoculation concentration on root-knot nematode disease of tomato: A total of 6 treatments were set up, including CK, BS fermentation stock solution, 2-fold dilution, 5-fold dilution, 10-fold dilution and 20-fold dilution treatment groups. 200ml of inoculation agent per plant when transplanting.

Inhibitory effect of BS inoculation dose on tomato root-knot nematode: AM fungal inoculation dose is 2000IPU/strain, BS inoculation fermentation stock solution, inoculation doses are (per plant): 0ml, 20ml, 50ml, 100ml, 200ml, 500ml respectively.

The inhibitory effect of BS inoculation frequency and inoculation timing on tomato root-knot nematode: the inoculation dose of AM fungus is 2000IPU/plant, the inoculation dose of BS inoculation fermentation stock solution is 200ml/plant, the inoculation frequency and inoculation timing are divided into 11 treatment groups: CK , transplanting, seedling stage, flowering and fruiting stage, fruiting stage, transplanting + seedling stage, transplanting + flowering and fruiting stage, transplanting + fruiting stage, transplanting + seedling stage + flowering and fruiting stage, transplanting + seedling stage + fruiting stage stage, transplanting + flowering and fruit setting stage + seedling stage.

(2) Test method

Each treatment group was inoculated with G.v bacterial agent 2000IPU when sowing. Sow two sterilized and germinated tomato seeds in the seedling soil premixed with AM bacterial agent, 2 plants per pot, and repeat 6 times. When the tomato seedlings grow to the 3-4 leaf stage, they are transplanted into flower pots, and at the same time, the roots are filled with the inoculum according to the BS inoculation concentration, inoculation dose and inoculation frequency. The rest are under normal management.

60 days after inoculation, the disease index and control effect of root-knot nematode disease incidence in each treatment group were determined.

(3) Results

The inhibitory effect of table 11BS fermented liquid concentration on tomato root-knot nematode

Inhibitory effect of table 12BS bacterial agent inoculation dosage on tomato root-knot nematode disease

Table 13 Inhibition effect of BS bacterial agent inoculation timing and inoculation frequency on tomato root-knot nematode disease

(3) Inhibition of tomato root-knot nematode disease inoculated with G.v+BS in protected areas

1. Experimental design

A total of 3 treatments were set up: control group (CK), thiazophosphine treatment group and G.v+BS treatment group. The G.v+BS treatment group was inserted with the G.v bacterial agent when sowing, and the BS fermentation stock solution was inserted when transplanted; when the thiazolylphosphine treatment group was transplanted, the thiazolylphosphine pesticide (active ingredient 10%, the manufacturer was Japan Ishihara Sangyo Co., Ltd., The drug effect is 4-5 months).

2. Selection of tomato protection areas

A tomato greenhouse with a serious incidence of root-knot nematode was selected. It is located in Dongcun Village, Xingcun Town, Haiyang City, Yantai City, Shandong Province (120°50 E, 36°16 N). The service life of the greenhouse is 13 years. stubble. The test period was from August 2013 to January 2014. The incidence of root-knot nematode on the last harvested tomato was relatively severe and common. The highest disease level reached level 5, and the incidence rate was about 75%.

3. Seedling cultivation and G.v inoculation

Tomato seeds were sterilized by 75% ethanol for 5 minutes and 2% NaClO for 20 minutes, and after germination, they were sown in 5×5 black hole trays, 1 plant/hole. The cultivation soil was a mixture of soil and seedling substrate (volume ratio 1:1). The G.v+BS treatment group was inoculated with 2000 IPU of G.v bacterial agent, and the control group and thiazophosphine treatment group were inoculated with an equal amount of sterilized mixed inoculum. normal management.

4. Colonization

The tomato seedlings at the 3-4 leaf stage were transplanted into tomato greenhouses with a plant spacing of 20 cm and a row spacing of 50 cm, with 100 plants per treatment. The G.v+BS treatment group was irrigated with 200ml/plant of BS fermentation stock solution at the first transplanting stage; the thiazophosphine pesticide was sprayed on the soil at a dose of 1500g/mu before transplanting in the thiazophosphine treatment group, and mixed with the upper soil Transplant tomato seedlings. After the BS fermented liquid infiltrates into the soil and dries up slightly, the furrows are irrigated with water. The rest are under normal management.

5. Sampling

Take the first sample at the seedling stage 15 days after planting, dig out the tomato root system and the soil around the root together, and try not to damage the root system. After sampling, the G.v+BS treatment group was inoculated with root irrigation for the second time at the seedling stage, and then inoculated with 100ml/plant after soil cultivation. 35 days after transplanting, take the second sampling at the flowering and fruit-setting period. After sampling, take the third time to irrigate the roots and insert 200ml of BS fermentation stock solution per plant. Take the third sampling at the fruiting period 75 days after transplanting.

6. Results

3.2.2 Inhibitory effect of G.v+BS and thiazophos on tomato root-knot nematode

G.v+BS and thiazophos have different effects on plant growth and development, and also have different control effects on tomato root-knot nematode. The root-knot nematode disease was the most serious in the CK control group, with the highest incidence rate of 84.38%, and the highest disease index was 0.448. The average number of root-knot nematodes per plant was 234.63/plant when sampling for 75 days; thiazophosphine pesticides can strongly inhibit root-knot nematode disease , only a few root knots were found in individual plants, and the incidence rate was less than 10%. The disease index was 0.053 and 0.038 at 35 days and 75 days after planting, and the control effects of three samplings were 100%, 85.24% and 91.52% respectively; G.v+ BS mixed inoculation treatment group can also effectively inhibit tomato root-knot nematode, the incidence rate is between 20-30%, the lowest disease index is 0.096, the number of root knots per plant is less than 40/plant, and the highest control effect is 78.57%.

Table 14 Number of root knots per plant of tomato root-knot nematode in different treatments (pieces)

Table 15 Different treatment tomato root-knot nematode disease incidence (%)

Table 16 Different treatment tomato root-knot nematode disease index (%)

Table 17 Different treatments of tomato root-knot nematode control effect (%)

Table 18 Tomato root length (cm) of different treatments

Table 19 Fresh weight of tomato roots in different treatments (g)

7. Hazards of thiazophosphine nematicide pesticides

Thiazophosphine is a commonly used nematicide in the world, which can prevent and treat root-knot nematodes, root rot (short body) nematodes, cyst nematodes, stem nematodes, etc. of various crops. The advantage is that the treatment efficiency is higher and the duration is longer. The main hazard is that thiazophos is absorbed by plants. In addition to killing various diseases and insect pests, it may have certain toxicity to plants and humans. Studies have shown that a certain dose of thiazophos may cause root length and root fresh weight to be significantly smaller than those of the control, and root vigor Low; animal experiments with rats as a model also showed that high doses of thiazophosphine lead to triglycerides, total protein, albumin, total bilirubin, creatinine, blood sugar, cholinesterase activity, etc. were significantly lower than those in the control group. This experiment also pointed out that the application of thiazophosphine at normal doses to the root system will inhibit the development of the tomato root system, resulting in a decrease in root length and root fresh weight, poor development of fibrous roots, and hindering the normal growth of plants. See Table 18-20.

Table 20 Fresh weight of lateral root of tomato in different treatments (g)

Claims (7)

1. a complex micro organism fungicide, is characterized in that, the activeconstituents of described microbiobacterial agent is made up of Glomus versiforme (Glomus versiforme, G.v) and subtilis (Bacillus subtilis, BS).

2. the biocontrol fungicide of the control tomato root-knot eelworm disease adopting complex micro organism fungicide according to claim 1 to prepare.

3. the application of biocontrol fungicide product described in claim 2 in control tomato root-knot eelworm.

4. adopt the method for biocontrol fungicide according to claim 2 control tomato root-knot eelworm disease, it is characterized in that, first, in the tomato seedling phase, in nursery soil, inoculate Glomus versiforme G.v; Again, in transplanting time, seedling stage and bloom the phase of bearing fruit, point inoculate described subtilis BS for 3 times.

5. the method for complex micro organism fungicide control tomato root-knot eelworm disease according to claim 4, it is characterized in that, the dosage of inoculation of described G.v is 1000-5000IPU/ strain; Described BS dosage of inoculation is fermented liquid 10 ⁹cfu/ml, 200-500ml/ strain.

6. the method for complex micro organism fungicide control tomato root-knot eelworm disease according to claim 5, it is characterized in that, the dosage of inoculation of described G.v is 2000IPU/ strain; Described BS dosage of inoculation is fermented liquid 10 ⁹cfu/ml, 200ml/ strain.

7. the method for complex micro organism fungicide control tomato root-knot eelworm disease according to claim 4, is characterized in that, described transplanting time is that tomato can Planting time when being developed to 3-4 leaf; Seedling stage is be developed to the 4-5 leaf stage after tomato field planting; Bloom the phase of bearing fruit be tomato be developed to one spend one fruit the phase.