A method for screening for resistance to olive root rot in rootstocks

Abstract

This invention belongs to the field of avocado disease resistance screening technology, specifically relating to a method for screening rootstocks resistant to avocado root rot, comprising the following steps: S1. Selecting plump, disease-free avocado seeds to be screened, disinfecting them, and then hydroponically cultivating them to obtain avocado hydroponic seedlings; S2. Activating and propagating the avocado root rot pathogen and preparing a mycelial suspension; S3. Rinsing the roots of the avocado hydroponic seedlings with distilled water and placing them in sterile hydroponic cups containing the mycelial suspension for cultivation and observation; S4. Grading and investigating the disease incidence of the avocado hydroponic seedlings and calculating the disease index (DI); S5. Classifying the resistance level according to the disease index (DI). This invention, by establishing a standardized hydroponic inoculation identification method, successfully overcomes the inherent defects of traditional methods, achieving significant progress in screening efficiency, result accuracy, and operational feasibility, providing efficient, reliable, and strong support for avocado root rot resistance breeding.

Description

Technical Field

[0001] This invention belongs to the field of avocado disease resistance screening technology, specifically relating to a method for screening rootstocks resistant to avocado root rot. Background Technology

[0002] Avocado root rot, also known as Phytophthora root rot, is caused by Phytophthora camphorata (… Phytophthora cinnamomi This pathogen infects the roots of avocados, causing them to rot and lose their ability to absorb water and nutrients. Above-ground symptoms include wilting, yellowing, and shedding of leaves, ultimately leading to the death of the entire plant. Root rot has caused devastating damage in avocado-producing regions worldwide, destroying orchards and resulting in enormous economic losses.

[0003] Currently, the main methods for controlling avocado root rot include chemical control and agricultural control. Chemical control uses fungicides (such as metalaxyl and phosphonates) for soil treatment or root drenching, which is costly and prone to pesticide residues and environmental pollution. Long-term use may lead to drug resistance in pathogens, and the effectiveness is unstable. Agricultural control methods, such as improving orchard drainage, using mulch, and avoiding over-irrigation, create an environment unfavorable to pathogen growth to achieve control. However, these methods can only mitigate the damage to a certain extent and cannot fundamentally solve the problem, especially in areas with high disease incidence. Utilizing disease-resistant rootstocks is recognized as the most economical, effective, and environmentally friendly control strategy. By selecting and grafting disease-resistant rootstocks, the plant's resistance to root rot can be fundamentally improved, enabling sustainable production.

[0004] Currently, the main methods for screening resistant rootstocks include field screening and indoor inoculation, but both have certain shortcomings: (1) Field screening method: This method has a long screening cycle. From seed germination and tree growth after grafting to resistance evaluation after natural infection or artificial inoculation in the field, the whole process takes several years. Environmental conditions are uncontrollable. Environmental factors such as soil moisture, temperature, microbial community and uneven distribution of pathogens in the field vary greatly, which seriously affects the accuracy and repeatability of the test results. The screening efficiency is low and it is impossible to achieve high-throughput rapid screening of a large number of candidate germplasm resources.

[0005] (2) Indoor inoculation method: The traditional indoor inoculation method uses soil-grown seedlings as experimental materials. The root growth of seedlings is affected by soil texture, and there are significant differences between individuals. The inoculation operation is difficult, and soil inoculation cannot ensure that the pathogens are in full and uniform contact with the roots of each seedling. The results are difficult to evaluate. Under soil cultivation conditions, it is impossible to directly observe and measure the root disease process without damaging the root system. Usually, the destructive scoring can only be done at the end of the experiment by observing the above-ground symptoms or digging up the roots, and the accuracy of the obtained data is insufficient.

[0006] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0007] The purpose of this invention is to provide a method for screening rootstocks resistant to root rot in avocados, so as to solve the problems existing in the prior art.

[0008] To achieve the above objectives, the present invention provides the following technical solution: A method for screening rootstocks resistant to root rot in avocado includes the following steps: S1. Select plump, disease-free avocado seeds for screening, disinfect them, and then cultivate them in water. Select avocado seedlings with 6-10 true leaves, vigorous growth, and well-developed and uniform root systems for inoculation experiments. S2. After activating and propagating the pathogen of avocado root rot, prepare a mycelial suspension; S3. After rinsing the roots of the avocado hydroponic seedlings with distilled water, the seedlings were placed in a sterile hydroponic cup containing mycelial suspension and cultured at 28°C. The growth of the avocado hydroponic seedlings was observed after 7 days. S4. Conduct a graded survey of disease incidence in hydroponic avocado seedlings and calculate the disease index DI. S5. Classify the resistance level according to the disease severity index (DI).

[0009] Furthermore, in step S2, the pathogen causing avocado root rot is *Phytophthora camphorata* (…). Phytophthora cinnamomi The mycelial suspension must be used within 2 hours.

[0010] Further, in step S2, the method for preparing the mycelial suspension is as follows: the avocado root rot pathogen is inoculated onto a 20% V8 solid medium, and after it has covered the petri dish, a 5mm diameter mycelial cake is taken; then 6 mycelial cakes are inoculated into a 20% V8 liquid medium, cultured in a constant temperature shaker at 28℃ for 7 days, and then filtered with sterile gauze; 10g of mycelium is taken into 300mL of sterile water and stirred with a stirrer to prepare a mycelial suspension.

[0011] Furthermore, in step S4, the grading criteria are as follows:

[0012] Furthermore, in step S4, the formula for calculating the disease index DI is as follows:

[0013] Furthermore, in step S5, the resistance grading criteria are as follows:

[0014] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention shortens the screening cycle to several weeks or months, greatly accelerating the breeding process. Moreover, the hydroponic system is easy to standardize and operate, and can process hundreds or thousands of rootstock materials in a limited space at the same time, realizing rapid initial screening of large-scale germplasm resources.

[0015] (2) The hydroponic inoculation of this invention is carried out in a controlled indoor environment, which effectively eliminates the interference of uncertain factors in the field such as soil and climate, making the identification results purer and more reliable. By directly immersing the roots in the bacterial suspension, the pathogen is fully, evenly and quantitatively contacted with the roots of each seedling, avoiding the heterogeneity of soil inoculation and ensuring the repeatability of the experiment and the comparability of the data. It can directly, in situ and dynamically observe the entire process of root system from infection to disease, providing an intuitive basis for accurately assessing the strength of disease resistance (such as resistance to invasion and resistance to spread).

[0016] (3) The cultivation, inoculation and observation process of hydroponic seedlings in this invention is standardized, easy to master and operate, and lowers the technical threshold. It saves a lot of land, manpower and time costs required for field trials, and indoor operations are also easier to manage.

[0017] In summary, this invention has successfully overcome the inherent defects of traditional methods by establishing a standardized hydroponic inoculation identification method, achieving significant progress in screening efficiency, result accuracy, and operational feasibility, and providing efficient, reliable, and powerful support for breeding avocados resistant to root rot. Attached Figure Description

[0018] Figure 1 The growth of Walter Hole and Dupuis avocados 7 days after inoculation; Explanation of key figure labels: Figure 1 In the diagram, A stands for Walter hole; B stands for Dupuis. Detailed Implementation

[0019] The technical solution of this invention patent will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention.

[0020] A method for screening rootstocks resistant to root rot in avocado includes the following steps: S1. Select the following avocado varieties as test subjects: Walter hole, Dupuis, Hass, Lisa, Fuerte, Bernecker, Duke7, Zijin, Tenglong, Guilong No. 2, Guiken Da No. 2, and Nanya A. Collect fresh avocado fruits, remove the seeds, and select plump and disease-free avocado seeds for screening. Soak the seeds in a 1% sodium hypochlorite solution for 5 minutes, then rinse them three times with sterile distilled water to remove surface bacteria. Place the sterilized seeds in hydroponic cups, add sterile water, and place them in a 28℃ constant temperature incubator for hydroponic cultivation. After about 6-8 weeks of cultivation, when the seedlings have grown 6-10 true leaves and developed a root system, they can be used for inoculation experiments. S2. The pathogen of avocado root rot, Phytophthora camphorata ( Phytophthora cinnamomi Inoculate onto 20% V8 solid medium and incubate at 28℃. Once the mycelium has fully colonized the petri dish (9cm in diameter), break it into 5mm diameter mycelial discs. Inoculate 6 mycelial discs into Erlenmeyer flasks containing 150mL of 20% V8 liquid medium and incubate at 120rpm in a shaker at 28℃. After 7 days, filter the mycelium with sterile gauze. Weigh 10g of mycelium into 300mL of sterile water and use a stirrer to prepare 5.0×10⁻⁶ mycelium. 4 CFU / mL mycelial suspension; The formula for 20% V8 solid culture medium is: 200mL V8 vegetable and fruit juice, 2g CaCO3, 20g agar, and 1000mL water; The formula for 20% V8 liquid culture medium is: 200mL V8 vegetable and fruit juice, 2g CaCO3, and 1000mL water; S3. Select robust avocado hydroponic seedlings with uniform root conditions and 6-10 true leaves. Gently rinse the roots with sterile distilled water. Place 300 mL of mycelial suspension in a sterile hydroponic cup (500 mL volume), completely immerse the roots of the avocado hydroponic seedlings in the mycelial suspension, and incubate in a 28℃ constant temperature greenhouse. Observe the growth of the avocado hydroponic seedlings after 7 days. Figure 1 ); S4. Conduct a graded survey of disease incidence in hydroponic avocado seedlings and calculate the disease index DI; The grading criteria are as follows:

[0021] The formula for calculating the Disease Index (DI) is as follows:

[0022] S5. Classify resistance levels based on the disease severity index (DI). The resistance grading criteria are as follows:

[0023] In this embodiment, 50 plants of each variety were inoculated, and the results of the screening of resistant rootstocks are shown in Table 1.

[0024] Table 1. Screening results of rootstocks resistant to root rot in different varieties of avocado Serial Number Variety Name Disease index Resistance classification 1 Walter hole 68.4 Sickness (S) 2 Dupuis 16.4 Disease resistance (R) 3 Hass 50.4 Sickness (S) 4 Lisa 35.6 MR (metastatic antimicrobial) 5 Fuerte 48.8 MR (metastatic antimicrobial) 6 Bernecker 28.8 Disease resistance (R) 7 Duke7 20.0 Disease resistance (R) 8 Zijin 36.0 MR (metastatic antimicrobial) 9 Tenglong 40.0 MR (metastatic antimicrobial) 10 Guiyan No. 2 52.8 Sickness (S) 11 Guiken No. 2 47.2 MR (metastatic antimicrobial) 12 South Asia A 33.6 MR (metastatic antimicrobial) Previous studies have shown that the Duke7 variety is susceptible to Phytophthora root rot, while the Hass variety is susceptible. As shown in Table 1, the test results of this application are consistent with these findings.

[0025] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A method for screening rootstocks resistant to root rot in avocado, characterized in that, Includes the following steps: S1. Select plump, disease-free avocado seeds for screening, disinfect them, and then cultivate them in water. Select avocado seedlings with 6-10 true leaves, vigorous growth, and well-developed and uniform root systems for inoculation experiments. S2. After activating and propagating the pathogen of avocado root rot, prepare a mycelial suspension; S3. After rinsing the roots of the avocado hydroponic seedlings with distilled water, the seedlings were placed in a sterile hydroponic cup containing mycelial suspension and cultured at 28℃. The growth of the avocado hydroponic seedlings was observed after 7 days. S4. Conduct a graded survey of disease incidence in hydroponic avocado seedlings and calculate the disease index DI; S5. Classify the resistance level according to the disease severity index (DI).

2. The method for screening rootstocks resistant to root rot of avocado according to claim 1, characterized in that, In step S2, the pathogen causing root rot in avocados is Phytophthora camphorata (… Phytophthora cinnamomi The mycelial suspension must be used within 2 hours.

3. The method for screening rootstocks resistant to root rot of avocado according to claim 1, characterized in that, In step S2, the method for preparing the mycelial suspension is as follows: the avocado root rot pathogen is inoculated onto a 20% V8 solid medium. After it has covered the petri dish, a 5mm diameter mycelial cake is taken. Then, 6 mycelial cakes are inoculated into a 20% V8 liquid medium and cultured in a constant temperature shaker at 28℃ for 7 days. After filtration with sterile gauze, 10g of mycelium is taken into 300mL of sterile water and stirred with a stirrer to prepare a mycelial suspension.

4. The method for screening rootstocks resistant to root rot of avocado according to claim 1, characterized in that, In step S4, the grading criteria are as follows: 。 5. The method for screening rootstocks resistant to root rot of avocado according to claim 1, characterized in that, In step S4, the formula for calculating the disease index DI is as follows: 。 6. The method for screening rootstocks resistant to root rot of avocado according to claim 1, characterized in that, In step S5, the resistance grading criteria are as follows: 。

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