Application of microsporum longipilum in enhancing the resistance to lodging and tillering ability of rice

Abstract

The application of Microbispora longispora in simultaneously enhancing the lodging resistance and tillering ability of rice belongs to the field of biological bacterial manure and rice planting technology. In order to solve the technical problems that the existing technology lacks an effective microbial technical solution capable of simultaneously solving the requirements of'strong stem' and 'tillering' of rice, the seed soaking and inoculation method cannot be compatible with the conventional seed treatment of bactericidal agents, the effective period is short, and the application effect of Microbispora in actual agricultural production is seriously restricted, the application of Microbispora longispora in simultaneously enhancing the lodging resistance and tillering ability of rice is provided, and specifically, the rice seedbed soil is treated by Microbispora longispora, more contact opportunities and time are given to Microbispora and rice roots, the conventional seed bactericide can be compatible, the lodging resistance and tillering ability of rice can be simultaneously enhanced, and the yield of rice is improved.

Description

Technical Field

[0001] This invention belongs to the field of bio-fertilizer and rice planting technology, specifically involving the application of *Microsporum longiflorum* in simultaneously enhancing the lodging resistance and tillering ability of rice. Background Technology

[0002] Rice is one of the most important food crops in my country and even the world. In high-yield rice cultivation practices, lodging and tillering are two key bottlenecks restricting the improvement of rice yield and quality. Lodging not only makes mechanical harvesting difficult and significantly reduces production efficiency, but also causes panicle sprouting and mold, resulting in serious yield loss and quality deterioration; while tillering, as a core agronomic trait that determines the number of effective panicles per unit area, directly determines the final yield of rice.

[0003] Currently, conventional strategies for addressing lodging in agricultural production mainly fall into two categories: First, relying on genetic improvement to breed lodging-resistant varieties with reduced plant height and enhanced stem strength. However, this strategy involves a long breeding cycle and high costs, and lodging resistance often exhibits genetic antagonism with other desirable agronomic traits, making it difficult to quickly meet production demands. Second, relying on agronomic measures, such as reasonable planting density, water control and field drying, and optimized nitrogen fertilizer management. However, these measures are often constrained by factors such as climate conditions and field management levels in practical application, resulting in unstable effects. Furthermore, excessive control of fertilizer and water may negatively impact tillering and panicle differentiation, creating a contradiction between yield increase and lodging resistance.

[0004] Current methods for promoting tillering mainly rely on the application of chemical fertilizers, especially nitrogen fertilizers. While sufficient nitrogen fertilizer can effectively stimulate tillering, excessive application can lead to excessive vegetative growth, increased ineffective tillering, poor ventilation and light penetration within the rice plant, exacerbating the risk of lodging, and causing a series of problems such as increased production costs, environmental pollution, and soil compaction. Therefore, finding a green and sustainable technological approach that can enhance rice's lodging resistance and effectively promote tillering has become a prominent and urgent problem to be solved in current rice production.

[0005] In recent years, the research and application of microbial fertilizers and plant growth-promoting bacteria have provided new directions for green crop production. Numerous reports have shown that certain beneficial plant microorganisms can promote crop growth through mechanisms such as nitrogen fixation, phosphorus solubilization, and growth hormone secretion. However, the effects of microorganisms are highly strain-specific, and current research and applications mostly focus on the single promoting effects of microorganisms on crop nutrient absorption, biomass accumulation, or stress resistance (such as drought resistance and salt tolerance).

[0006] genus *Xanthomonas* ( Parametarhizium *Spp.* is a new genus of fungi isolated and established in 2021 from forest litter in Northeast China, including *Xanthomonas xinganensis* (spp.). P. hingganense ) and Microsporum longiflorum ( P. changbaienseTwo new fungal species were named in the article (Gao S, Meng W, Zhang L, Yue Q, Zheng X and Xu L (2021)). Parametarhizium ( Clavicipitaceae Before the publication of *Microsporum changbaiense* gen. nov. With Two New Species as a Potential Biocontrol Agent Isolated From Forest Litters in Northeast China. Front. Microbiol. 12:627744. doi: 10.3389 / fmicb.2021.627744, it was previously known as *Metarhizium anisopliae*. Metarhizium changbaiensis ), Xingan yellow microsporidium was formerly known as Xingan green muscardine ( Metarhizium hingganensis ).

[0007] Studies have found that *Xanthomonas* fungi possess insecticidal and anti-plant pathogenic activity, and can promote rice seedling growth. However, it remains unclear whether *Xanthomonas* fungi can improve lodging resistance and tillering ability in rice.

[0008] It is particularly important to note that crop lodging resistance is a complex and comprehensive trait comprised of multiple factors, including stem mechanical strength and plant morphology, while tillering is a developmental process precisely regulated by endogenous hormones. Currently, research on microbial agents largely focuses on general functions such as promoting growth and stress resistance; there are no in-depth reports or product developments demonstrating that the same strain can simultaneously and synergistically act on both lodging resistance and tillering—two key physiological processes in rice. This field lacks an effective microbial technology solution that can comprehensively address the needs for both "strong stems" and "increased tillering" in rice.

[0009] Furthermore, in-depth research reveals significant limitations in the application of *Xanthomonas xinganensis* and *Xanthomonas longiflora* in existing technologies. In applications using these two fungi to promote the growth of mung bean and rice seedlings, the method employed is seed soaking with a fungal suspension. While this method demonstrates some growth-promoting effects under laboratory conditions, it faces numerous problems in practical field applications. Firstly, modern rice cultivation commonly employs chemical fungicides (primarily antifungicides) for seed soaking or coating before sowing to control seed-borne and soil-borne diseases. These chemical fungicides can kill or inhibit beneficial microorganisms, including *Xanthomonas xinganensis*, thereby reducing the beneficial effects of *Xanthomonas xinganensis* fungi. However, if seed treatment (soaking or coating) is not performed using chemical fungicides, and *Xanthomonas xinganensis* fungi are used alone, the limited antibacterial spectrum of *Xanthomonas xinganensis* fungi increases the risk of plant diseases. Therefore, a method for applying *Xanthomonas xinganensis* fungi that is compatible with modern rice seedling raising techniques (chemical fungicide seed treatment) is needed. Secondly, *Xanthomonas* fungi colonize the roots of rice. Seed soaking or coating, limited by seed size and treatment time, restricts the fungi's contact with rice roots. However, lodging resistance and tillering require the continuous influence of beneficial microorganisms on rice, and the effective period of seed soaking is relatively short, making it difficult to guarantee the sustained effect of *Xanthomonas* on rice. This severely limits the multifunctional potential of *Xanthomonas*. Furthermore, unlike other field crops, rice is mostly not directly sown but rather raised in shallow seedbeds and transplanted. Therefore, it is worthwhile to develop new methods specifically tailored to current seedling raising techniques. In summary, the seed soaking inoculation method, due to its incompatibility with conventional seed coating agronomy and its short effective period, severely restricts the application effect of *Xanthomonas* in actual agricultural production, greatly limiting the promotion value and application prospects of *Xanthomonas* technology.

[0010] Therefore, developing a novel microbial application technology compatible with modern rice cultivation methods and overcoming the limitations of seed treatment to enhance the mechanical strength of rice stems and promote effective tillering is of great significance for reducing reliance on chemical fertilizers, lowering the risk of lodging, and achieving high-yield, high-quality, and green rice production. Summary of the Invention

[0011] To address the lack of an effective microbial technology solution that can comprehensively address the needs of strong stems and increased tillering in rice, and the technical problems such as the incompatibility of seed soaking and inoculation methods with conventional rice seed fungicides and their short effective period, which severely restrict the application of *Microsporum xanthophyte* in actual agricultural production, this invention provides the application of *Microsporum xanthophyte* fungi in simultaneously enhancing the lodging resistance and tillering ability of rice. Specifically, a spore suspension of *Microsporum xanthophyte* is sprayed onto the seedbed soil before or after sowing to simultaneously enhance the lodging resistance and tillering ability of rice, thereby increasing rice yield. This method is compatible with modern rice seedling raising methods, and the use of rice seed fungicides and seedbed herbicides does not affect the colonization of *Microsporum xanthophyte*.

[0012] To solve the above-mentioned technical problems and achieve the corresponding technical effects, the present invention provides the following technical solution:

[0013] The first objective of this invention is to provide *Microsporum longum* (… Parametarhizium changbaiense The application of *Microsporum longiflorum* in simultaneously enhancing the lodging resistance and tillering ability of rice involves using it to prepare a bio-fertilizer that simultaneously enhances the lodging resistance and promotes tillering ability of rice.

[0014] In one embodiment of the present invention, enhancing the lodging resistance of rice refers to increasing the maximum bending force, maximum flexural strength, stem wall thickness, and reducing the length of the second internode.

[0015] The second objective of this invention is to provide *Microsporum longum* (… Parametarhizium changbaiense The application of *Microsporum longiflorum* in simultaneously enhancing the lodging resistance and tillering ability of rice involves using it to prepare a microbial agent that simultaneously enhances the lodging resistance and promotes the tillering ability of rice.

[0016] In one embodiment of the present invention, enhancing the lodging resistance of rice refers to increasing the maximum bending force, maximum flexural strength, stem wall thickness, and reducing the length of the second internode.

[0017] The third objective of this invention is to provide a seedbed treatment method that simultaneously enhances the lodging resistance and tillering ability of rice. This seedbed treatment method involves applying *Microsporum longiflorum* (a type of fungus) to the seedbed soil during the preparation of rice seedbed soil. Parametarhizium changbaiense The spore suspension is then used for sowing.

[0018] In one embodiment of the present invention, enhancing the lodging resistance of rice refers to increasing the maximum bending force, maximum flexural strength, stem wall thickness, and reducing the length of the second internode.

[0019] In one embodiment of the present invention, the amount of the spore suspension of *Microsporum longum* is 1 × 10⁻⁶ per 9-inch seedling tray.9 One spore.

[0020] The fourth objective of this invention is to provide a seedbed treatment method that simultaneously enhances the lodging resistance and tillering ability of rice. The seedbed treatment method involves uniformly spraying a suspension of spores of Microsporidium longiflorum onto the seedbed covering soil after rice seedlings have been sown but before they emerge.

[0021] In one embodiment of the present invention, enhancing the lodging resistance of rice refers to increasing the maximum bending force, maximum flexural strength, stem wall thickness, and reducing the length of the second internode.

[0022] In one embodiment of the present invention, the amount of the spore suspension of *Microsporum longum* is 2 × 10⁻⁶ per 9-inch seedling tray. 9 One spore.

[0023] The beneficial effects of this invention are:

[0024] This invention investigates the effects of adding a suspension of *Microspora longiflora* spores to seedbed soil on lodging resistance and tillering ability in rice. The results show that, under similar plant height conditions, the *Microspora longiflora* spore suspension significantly improved lodging resistance in rice. Specifically, compared to the control group, it significantly increased maximum bending force, maximum flexural strength, and stem thickness, while decreasing the length of the second internode and promoting lignification. Furthermore, compared to the control group (effective tiller number per hill: 19.6±0.85; grain number per panicle: 87.94±10.5), the *Microspora longiflora* treatment group significantly increased the effective tiller number per hill (22.3±0.54) and the grain number per panicle (95.69±11.19).

[0025] This invention involves spraying a suspension of *Microsporum longiflorum* spores onto the seedbed after rice sowing to study its effects on lodging resistance and tillering ability in rice. The results showed that 40 days after spraying, the average number of tillers in the *Microsporum longiflorum* treatment group (2.40±0.83) was significantly higher than that in the control group (1.40±0.74). At maturity, the maximum bending force in the *Microsporum longiflorum* treatment group (19.45±4.29 Newtons) was significantly higher than that in the control group (12.45±3.14 Newtons). At harvest, the number of effective tillers in the *Microsporum longiflorum* treatment group (25.20±5.81) was significantly higher than that in the control group (19.95±4.80).

[0026] This invention demonstrates through experiments that adding a spore suspension of *Microsporum longiflorum* to the rice seedbed soil, or spraying a spore suspension of *Microsporum longiflorum* onto the seedbed soil after rice sowing, can significantly improve the lodging resistance and tillering ability of rice, thereby increasing rice yield. Furthermore, both seedbed treatment methods provided by this invention are compatible with modern rice seedling raising methods, ensuring that the use of rice seed fungicides and seedbed herbicides does not affect the colonization of *Microsporum longiflorum*. Attached Figure Description

[0027] Figure 1 This is a graph showing the statistical results of rice plant height in Example 1; where PC represents the *Microsporidium longiflorum* treatment group.

[0028] Figure 2 This is a statistical result graph of the maximum bending force of rice in Example 1; where PC represents the *Microsporidium leucopsis* treatment group;

[0029] Figure 3 This is a graph showing the statistical results of the maximum bending resistance of rice in Example 1; where PC represents the *Microsporidium leucopsis* treatment group;

[0030] Figure 4 This is a statistical result graph of the rice stem wall thickness in Example 1; where PC represents the *Microsporidium longiflorum* treatment group;

[0031] Figure 5 This is a statistical result graph of the second internode length of rice in Example 1; where PC represents the *Microsporidium longiflorum* treatment group;

[0032] Figure 6 The image shows lodging resistance in rice fields in Example 1; where A is the control group and B is the group treated with Microsporidium chrysogenum.

[0033] Figure 7 The image shows the staining of lignified cells in the rice stem in Example 1; A is the control group, and B is the group treated with Microsporidium leuciscus. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be noted that the embodiments mentioned below are only for explaining the invention and are not intended to limit the scope of the invention. The embodiments mentioned below are only some embodiments of the invention, not all embodiments. Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the objectives of the invention. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of this invention to realize and apply the technology of this invention. In the art, embodiments obtained by other those skilled in the art without creative effort are all protected by this invention.

[0035] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials, reagents, culture media and instruments used are all conventional materials, reagents, culture media and instruments in the art, which can be obtained by those skilled in the art through commercial channels.

[0036] The rice seeds used in this invention are as follows:

[0037] The rice used in Example 1 ( Oryza sativa L.) The seed variety is Hajingdao No. 2 (Approval No.: Heishendao 2014017), and the rice used in Example 2 is ( Oryza sativa L.) The seed variety is Daohuaxiang No. 2 (a common variety of Wuchang Daohuaxiang). Both of the above rice seed varieties are well-known rice varieties in this field and were donated by the Harbin Academy of Agricultural Sciences.

[0038] The microorganisms used in this invention are as follows:

[0039] Microsporidium longiflorum ( Parametarhizium changbaiense This is disclosed in Chinese Patent Application No. 202210962001.9, entitled "Application of a Microsporum fungus in promoting plant growth and improving plant stress resistance." (Gao S, Meng W, Zhang L, Yue Q, Zheng X and Xu L (2021)) Parametarhizium ( ClavicipitaceaeBefore the publication of *Microsporum changbaiense* gen. nov. With Two New Species as a Potential Biocontrol Agent Isolated From Forest Litters in Northeast China. Front. Microbiol. 12:627744. doi: 10.3389 / fmicb.2021.627744, it was previously known as *Metarhizium anisopliae*. Metarhizium changbaiensis The sample is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 19143 and deposit date of December 5, 2019.

[0040] Culture and preparation of spore suspension of *Microspora longiflora*:

[0041] *Microsporum longiflorum* was inoculated onto 9 cm PDA plates and incubated at 25°C for 14 days. Sterile 0.05% Tween 20 solution was added, and spores were scraped off. The spore suspension was filtered through sterile medical gauze (to remove hyphae). The spore concentration was then determined using a microscope and a hemocytometer, and the spore suspension was diluted to a concentration of 1 × 10⁻⁶ spores. 7 Quantity / mL, for later use.

[0042] Example 1: Effects of adding *Microsporum longiflorum* to seedbed soil on lodging resistance and tillering ability of rice.

[0043] Using a spore suspension of Microsporidium longiflorum (concentration of 1×10⁻⁶) 7 Treat the seedbed soil with 1×10⁻⁶ cells / mL, using 1×10⁻⁶ cells / mL per 9-inch seedling tray. 9 Rice seeds with plump grains and no disease spots were treated with fungicides (thiram or tebuconazole) and sown in seedbed soil pretreated with *Microspora leucovora*. Butachlor was used as the pre-emergence herbicide for seedbed application. Alternatively, rice seeds with plump grains and no disease spots were treated with fungicides (thiram or tebuconazole) and sown in seedbed soil not pre-treated with *Microspora leucovora*. Butachlor was used as the pre-emergence herbicide for seedbed application, serving as a blank control group.

[0044] 1. Rice lodging resistance test

[0045] After rice enters reproductive growth and before maturity, 30 to 60 rice plants are selected in the rice field to measure the maximum bending force, maximum flexural strength, second internode length, and stem wall thickness. The degree of lignification of the rice stem is also observed using a stereomicroscope.

[0046] Method for measuring maximum bending force:

[0047] Using the Top Cloud Agriculture YYD-1A plant stem strength tester, with the rice stem bending test head adapted, the rice stem was slowly bent to the preset angle (30°) according to the instrument's instructions. Then, the maximum bending force of the rice stem was measured in Newtons (N). After the value stabilized, the data was recorded.

[0048] Methods for measuring maximum bending strength, second internode length, and stem wall thickness:

[0049] The test site is located at the base of the rice plant (near the root), between the second and third nodes (the second internode). Adjust the height of the needle tip on the YYD-1A measuring instrument so that the needle tip is vertically aligned with the midpoint of the second internode. Following the instrument's instructions, control the needle tip to move downwards until the needle tip completely penetrates the rice stem. After the reading stabilizes, read and record the maximum bending strength value in Newtons (N). Measure the length of the second internode and the stem wall thickness. Cut a 2-3 cm section from the middle of the second internode, remove the pith, and measure the stem wall thickness using calipers.

[0050] Observation of the degree of lignification in rice stems:

[0051] The entire length of the second elongated internode of the main stem was cut off. After removing epidermal impurities, a 1 cm segment was taken from the same location 2-3 cm above the base as a fixed sampling point. The segments were then hand-sectioned and stained with phloroglucinol. The degree of lignification of the rice stem was observed using a stereomicroscope.

[0052] The measurement results of lodging resistance indicators show that, under the condition that the plant height is basically the same ( Figure 1 Microsporum longiflorum can enhance the lodging resistance of rice, specifically by significantly increasing maximum bending force, maximum flexural strength, and stem thickness. Figure 2 , Figure 3 , Figure 4 ), and significantly reduced the length of the second intersegment ( Figure 5 ). Figure 6 The field treatment with *Microsporum simulans* demonstrated its anti-lodging effect. Figure 7 The results of staining lignified cells of Microsporidium longiflorum were shown, and the degree of lignification was better than that of the control group.

[0053] 2. Rice tillering ability test

[0054] After rice harvest, 75 hills were selected for each treatment, and the number of effective panicles per hill (average) and the number of filled grains per panicle (average) were counted. Table 1 shows that the number of effective panicles per hill in the control group and the *Microsporum simonii* treatment group were 19.6±0.85 and 22.3±0.54, respectively. Compared with the control group, *Microsporum simonii* significantly increased the number of effective panicles (effective tillers). The number of filled grains per hill in the control group and the *Microsporum simonii* treatment group were 87.94±10.5 and 95.69±11.19, respectively. Compared with the control group, *Microsporum simonii* significantly increased the number of effective filled grains.

[0055] The above results indicate that adding *Microspora longiflora* to the seedbed soil can significantly improve rice's lodging resistance, effective panicle number (effective tiller number), and number of grains per panicle.

[0056] Table 1. Statistical results of the number of effective panicles (effective tillers) per hill and the number of filled grains per panicle in each treatment group.

[0057]

[0058] Example 2: Effects of spraying *Microsporum longiflorum* on rice lodging resistance and tillering ability after sowing and covering the seedbed with soil.

[0059] Select plump rice seeds without disease spots, treat them with fungicides (thiram, tebuconazole), and sow them in the seedbed soil. After sowing and before emergence, evenly spray a suspension of *Microspora leucocephala* spores onto the seedbed soil, applying 2 × 10⁻⁶ spores per 9-inch seedling tray. 9 1 spore. Pre-emergence herbicides (butachlor and sulfadiazine) were applied to the seedbed. The treatment without herbicide application served as the control group.

[0060] On day 40 after spraying the microbial agent, 15 rice plants were randomly selected from each treatment to investigate the average number of tillers. The average number of tillers in the control group was 1.40±0.74, and the average number of tillers in the *Microsporidium chrysogenum* treatment group was 2.40±0.83.

[0061] Organic farming methods were adopted in the Honda stage, and no chemical fertilizers were used. When the rice reached maturity (96 days after spraying the fungicide), the maximum bending force of the rice in the field was measured using instruments: the control group was 12.45±3.14 Newtons, and the treatment group with Microsporum longiflorum was 19.45±4.29 Newtons, which was significantly higher than the control.

[0062] At rice harvest, the average number of effective tillers (effective panicles) per hill was calculated: 19.95±4.80 in the control group and 25.20±5.81 in the group treated with Microsporidium chrysogenum, which was significantly higher than the control.

[0063] The above results indicate that spraying *Microsporum longiflorum* on the seedbed soil after rice sowing can significantly improve both the lodging resistance and the number of effective panicles (effective tillers) of rice.

[0064] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be defined by the claims.

Claims

1. Microsporum longum ( Parametarhizium changbaiense The application of spore suspension of ) in simultaneously enhancing lodging resistance and tillering ability of rice is characterized by, The application involves using a spore suspension of *Microspora longiflora* to prepare a bio-fertilizer that simultaneously enhances rice's lodging resistance and promotes tillering. The spore suspension is applied in the following ways: when preparing seedbed soil for rice seedling cultivation, the spore suspension of *Microspora longiflora* is applied to the seedbed soil before sowing; or after rice seedlings have been sown in the seedbed but before emergence, the spore suspension of *Microspora longiflora* is evenly sprayed onto the seedbed covering soil.

2. The application according to claim 1, characterized in that, Enhancing the lodging resistance of rice refers to increasing the maximum bending force, maximum flexural strength, stem wall thickness, and reducing the length of the second internode.

3. Microsporidium longiflorum ( Parametarhizium changbaiense The application of spore suspension of ) in simultaneously enhancing lodging resistance and tillering ability of rice is characterized by, The application involves using a spore suspension of *Microsporum longiflorum* to prepare a microbial agent that simultaneously enhances rice's lodging resistance and promotes tillering. The spore suspension is applied in the following ways: when preparing seedbed soil for rice seedling cultivation, the spore suspension of *Microsporum longiflorum* is applied to the seedbed soil before sowing; or after rice seedlings have been sown in the seedbed but before emergence, the spore suspension of *Microsporum longiflorum* is evenly sprayed onto the seedbed covering soil.

4. The application according to claim 3, characterized in that, Enhancing the lodging resistance of rice refers to increasing the maximum bending force, maximum flexural strength, stem wall thickness, and reducing the length of the second internode.

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