Leptospora cathayensis, fungicide and application of Leptospora cathayensis

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a Beiribospora cathayensis, a fungicide and application of the Beiribospora cathayensis. The Leptospora cathayensis is F3-31 and is preserved in the General Microbiological Culture Collection Center of the China Committee for Culture Collection of Microorganisms on December 18, 2024, and the preservation number of the Leptospora cathayensis is CGMCC (China General Microbiological Culture Collection Center) NO.33119. The screened Leptospora cathayensis has a good antagonistic effect on tobacco fusarium oxysporum and has a relatively strong inhibition effect on various common tobacco pathogenic fungi, and results of a plate confrontation method show that the average control effect of the bacterial strain on the tobacco fusarium oxysporum is 67.3%, and the bacterial strain has a good application prospect. The compound has a relatively strong inhibition effect on various common tobacco pathogenic fungi.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a type of *Cathaya argyrophylla*, its inoculant, and its applications. Background Technology

[0002] Tobacco root rot is a significant disease in tobacco cultivation, causing poor plant growth, yellowing leaves, and root rot. In severe cases, it can even lead to the death of the entire plant, seriously affecting tobacco yield and quality. To effectively control this disease, accurate identification of the pathogen is necessary. Currently, *Fusarium oxysporum* has been isolated and identified. Foxysporum Schlecht Fusarium solani, Fusarium semi-nakedense Fsemitectum It was clarified that all three types of Fusarium can infect tobacco. Fusarium infection in tobacco causes tobacco Fusarium root rot, a significant and devastating soil-borne disease that causes substantial economic losses to the tobacco industry. Symptoms become more pronounced during the plant's growth and development stages after infection, and once infected, plants are difficult to recover, resulting in significant losses for producers.

[0003] In recent years, due to adjustments in tobacco planting area, changes in farming systems, and tobacco varieties, tobacco root rot has shown a trend of increasing severity. Traditional tobacco cultivation methods rely heavily on chemical agents for disease control, which not only increases production costs but also poses potential threats to the environment and human health. Therefore, finding and developing microorganisms to control root rot is of great significance for improving tobacco yield and quality, reducing the use of chemical agents, and protecting the environment and human health. Currently, *Kitasatospora* sp. is used to promote plant growth, control eucalyptus bacterial wilt, and produce ε-polylysine, among other things. Microorganisms used to control tobacco root rot include *Streptomyces*, *Bacillus*, and *Pseudomonas*, but *Kitasatospora* is not yet available for controlling tobacco root rot. Summary of the Invention

[0004] To address the above problems, this invention provides *Cathaya argyrophylla*, its inoculant, and its applications.

[0005] A species of *Cinnamomum cassia*, specifically F3-31, is classified and named... Kitasatospora cathayae It was deposited on December 18, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.33119.

[0006] The *Cryptotympanyrum* strain of this invention exhibits good antagonistic activity against *Fusarium oxysporum* and strong inhibitory effects against various common tobacco pathogenic fungi, such as *Phytophthora nicotinae*, *Rhizoctonia solani*, and *Usnea iris*. The average control efficacy of this strain against *Fusarium oxysporum* is 67.3%, and it has a strong inhibitory effect against various common tobacco pathogenic fungi.

[0007] A microbial agent prepared from the aforementioned *Cathaya argyrophylla*.

[0008] Preferably, the preparation method of the bacterial agent is as follows: placing the *Cinnamomum cassia* in LB liquid medium and culturing it at 28°C for 48 hours to obtain the bacterial stock solution, centrifuging the bacterial stock solution, and then filtering the supernatant after sterilization.

[0009] The application of the aforementioned *Cathaya argyrophylla* or the aforementioned inoculant in the prevention and control of tobacco root rot, tobacco black shank disease, tobacco root black rot, and tobacco floating seedling root rot.

[0010] Preferably, the tobacco root rot is Fusarium root rot.

[0011] Preferably, the roots of tobacco seedlings are soaked in a suspension of *Cathaya argyrophylla* before planting to prevent *Fusarium* root rot in tobacco.

[0012] Preferably, after planting, the tobacco seedlings are treated with a suspension of the aforementioned *Cathaya argyrophylla* to prevent *Fusarium* root rot, with 20 mL applied to each seedling.

[0013] The application of *Cathaya argyrophylla* in the prevention and control of pathogenic fungi.

[0014] Preferably, the pathogenic fungi include Fusarium oxysporum, Phytophthora nicotineum, Rhizoctonia solani, and Mycotomyces iris.

[0015] The application of *Cathaya argyrophylla* in improving tobacco agronomic traits.

[0016] Preferably, the improved tobacco agronomic traits are any one of the following: plant height, leaf area, aboveground fresh weight, root fresh weight, root length, root surface area, and root volume.

[0017] Compared with the prior art, the advantages of the present invention are: The *Cinnamomum cassia* strain screened in this invention exhibits good antagonistic activity against *Fusarium oxysporum* and strong inhibitory effects against a variety of common tobacco pathogenic fungi. Results from the plate confrontation method show that this strain has an average control efficacy of 67.3% against *Fusarium oxysporum* and strong inhibitory effects against a variety of common tobacco pathogenic fungi.

[0018] The *Cathaya argyrophylla* strain screened in this invention exhibits good potted plant control efficacy against Fusarium root rot and good biocontrol and growth-promoting effects. Its average control efficacy against *Fusarium oxysporum* root rot in tobacco is 67.3%, making this invention an effective means of controlling *Fusarium oxysporum* root rot in tobacco and promoting its growth. Attached Figure Description

[0019] Figure 1 The image shows the antibacterial activity of *Cathaya argyrophylla* F3-31.

[0020] Figure 2 The diagram shows the antibacterial activity of fermentation filtrate of *Cathaya argyrophylla* F3-31. A represents the treatment without fermentation filtrate, and B represents the treatment with fermentation filtrate.

[0021] Figure 3 The phylogenetic tree of the antagonistic bacteria of the present invention is constructed based on Hsp70 rDNA.

[0022] Figure 4 The images show the effects of growth promotion, where A represents the result of CK treatment and B represents the result of F3-31 treatment. Detailed Implementation

[0023] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods.

[0024] Example 1 In May 2024, samples were collected from tobacco fields in Kuandian Manchu Autonomous County, Dandong City, Liaoning Province. A five-point sampling method was used, with each point spaced at least 10 meters apart. Three healthy plants were collected at each point. The roots of the tobacco plants and the rhizosphere soil were placed in sterile self-sealing bags. The plant species and time of sampling were recorded, and the samples were transported at low temperature to the laboratory for isolation of dominant biocontrol bacteria.

[0025] Collect soil from the rhizosphere of tobacco plants, removing large particles, and air-dry for 2 days. Weigh 5g of soil and add it to 95mL of sterile water, bringing the volume to 100mL. Mix thoroughly by shaking on a shaker at 28℃ and 160r / min for 30 minutes. After standing at room temperature for 10 minutes, collect the supernatant and serially dilute it 10 times with sterile water. -4 —10 -6 (bacteria) and 10 -5 —10 -7(Actinomycetes) Take 100 μL of each gradient dilution and spread it evenly on LB agar plates and Gao's No. 1 medium containing potassium dichromate (330 μL of potassium dichromate per 100 mL of Gao's No. 1 medium). Incubate at 28℃ for 5-7 days. Select strains with typical actinomycete colony characteristics and purify single colonies by streak plate method. After purification 3 times, number the purified strains and store 25% glycerol tubes in a -80℃ freezer.

[0026] Example 2 Antagonistic strains were screened using the plate confrontation culture method with Fusarium oxysporum as the target pathogen, and the inhibition rate was calculated.

[0027] The isolated bacteria were streaked vertically onto both sides of a PDA culture dish 2.5 cm from the center using a stinger. The dish was incubated at 28°C for 24 hours. After 5 days of incubation, the target pathogens were collected, and bacterial pellets were punched at the edge of the colony using a 5 mm diameter punch. These pellets were then inoculated into the center of a PDA plate inoculated with Fusarium oxysporum. A fungal plate without antagonistic bacteria was used as a control. The plates were incubated at 28°C. The inhibitory effect of each biocontrol bacterium and actinomycete on Fusarium oxysporum was measured, and the inhibition rate was determined.

[0028] Ninety bacterial strains were isolated from 82 soil samples. The strain F3-31, which exhibited the best antagonistic effect, was selected through the plate confrontation method. This strain was isolated from the rhizosphere soil of healthy tobacco plants in Kuandian Manchu Autonomous County, Dandong City. As shown in Table 2, the plate confrontation method results indicated that F3-31 had an average control efficacy of 67.3% against *Fusarium oxysporum* sp. var. *tobacco*. Figure 1 As shown.

[0029] Table 2: Antibacterial effect of biocontrol bacteria against Fusarium oxysporum tobaccoides Example 3 Antagonistic bacterial strain F3-31, effective against Fusarium oxysporum, was screened and further identified. Referring to Bergey's Manual of Bacteriological Identification, the antagonistic strain was streaked into LB medium and incubated at 30°C for 2-3 days. Single colony morphology was observed, and Gram staining and spore staining were performed. Physiological and biochemical indicators, including starch, glucose, lactose, sucrose, D-mannitol, OF test, gelatin hydrolysis, VP test, and methyl red test, were measured using micro-biochemical reaction tubes. Single colonies of the antagonistic strain were inoculated into LB medium and incubated at 32°C and 180 rpm for 48 h. Bacterial cells were collected, bacterial DNA was extracted, and DNA quality was assessed by agarose gel electrophoresis. The Hsp70 gene primers Hsp70-U7F (5'-CGTGCAGTCGGTATCGACCTCGGBACVACBAACTC-3', SEQ ID NO.1) and Hsp70-1326R (5'-CGATGCCGTTGGCGTCGATGTCGAASGHSACCTCGA-3', SEQ ID NO.2) were used to amplify the Hsp70r DNA of F3-31. In these primers, B represents the three bases other than A (C, G, T), V represents the three bases other than G (A, C, T), S represents C or G, and H represents the three bases other than T (A, C, G). The PCR reaction volume was 50 µL. The Hsp70r DNA gene sequence was aligned with all sequences in the database using the Basic Local Alignment Search (BLAST) tool from the National Center for Biotechnology Information (NCBI). A phylogenetic tree was constructed using MEGA7 software (https: / / www.megasoftware.net / ).

[0030] As shown in Table 1, data processing was performed using Excel and DPS 7.05 software, and the analysis of variance was performed using Duncan's new multiple range method.

[0031] Table 1: Detection Primer Information according to Figure 3 As shown, BLAST results indicate that the Hsp70 gene sequence of strain F3-31 is similar to that of *Cathaya argyrophylla* (…). Kitasatospora cathayae The sequence similarity between F3-31 and F4-31 reached 99%, and they were located in the same evolutionary branch. Therefore, F3-31 was identified as *Cryptospira crassa*. Kitasatospora cathayae ).

[0032] Example 4 The antibacterial activity of antagonistic bacterial fermentation filtrate was determined by preparing antagonistic bacterial fermentation filtrate, testing the antibacterial effect of biocontrol bacterial fermentation filtrate using the plate perforation method, and determining the effect of antagonistic bacterial fermentation filtrate on pathogenic spore germination.

[0033] Preparation of antagonistic bacterial fermentation filtrate: One loop of the LB slant culture strain was inoculated into a 250 mL Erlenmeyer flask containing 150 mL of LB liquid medium. The culture was carried out at 28 °C with shaking at 180 r / min for 48 h to obtain the stock solution. The bacterial solution obtained above was centrifuged at 12000 r / min for 10 min, and the supernatant was collected. The supernatant was filtered twice with a bacterial filter (22 μm) to obtain sterile fermentation filtrate.

[0034] The antibacterial effect of biocontrol bacteria fermentation filtrate was tested using the plate perforation method. Two symmetrical holes were punched 2.5 cm from the center of the plate, and 200 L of fermentation filtrate (9 mm in diameter) was injected into each hole. A sterile blank LB liquid medium was used as a control. After the fermentation broth was completely absorbed by the medium, a 5 mm diameter pathogenic bacteria block was inoculated into the center of the medium. Each treatment was repeated three times. Conidia of the pathogen were collected, and the spore concentration was adjusted to 1 × 10⁻⁶. 7 Spores / mL were mixed with equal volumes of fermentation filtrate from four antagonistic strains and co-cultured at 25°C for 24 h. Spore germination was observed under an optical microscope. Treatment with sterile LB liquid medium filtrate served as a control. Each treatment was repeated three times.

[0035] The measurement results are as follows Figure 2 The results showed that the fermentation filtrate of F3-31 had a significant inhibitory effect on the mycelial growth of the target pathogen Fusarium oxysporum (P<0.05), with an inhibition rate of 67.30%, which was comparable to the inhibition rate of live bacteria.

[0036] Example 5 Determination of the potted plant control efficacy of antagonistic bacteria against Fusarium root rot and the growth-promoting effect of biocontrol bacteria.

[0037] To investigate the potted plant control efficacy of antagonistic bacteria against Fusarium oxysporum root rot in tobacco and the growth-promoting effect of biocontrol bacteria, this invention conducted relevant experiments.

[0038] Healthy and plump K326 tobacco seeds were selected for the experiment and cultivated using the floating seedling method until the large cross stage (including 3-4 true leaves). During this period, healthy tobacco seedlings of uniform size were selected with sterile tweezers and the substrate at the roots of the seedlings was gently rinsed off with sterile water.

[0039] Preparation of antagonistic bacterial suspension: The tested antagonistic bacteria were first inoculated into LB liquid medium and cultured at 28–30°C with shaking at 150–200 rpm until the logarithmic growth phase. The bacterial suspension was then centrifuged at 5000–8000 rpm for 10–15 min to collect the bacterial cells. The cells were washed 2–3 times with sterile physiological saline or phosphate buffer to remove residual culture medium. The cells were then resuspended in sterile solution, and the concentration was adjusted to 10 using a hemocytometer or spectrophotometer (measuring absorbance at 600 nm). 8~10 9 CFU / mL yields the antagonistic bacterial suspension tested.

[0040] Tobacco seedlings were soaked in the antagonistic bacterial suspension for 30 minutes, then transplanted into sterile pots (85×70mm) containing a sterile substrate of nutrient soil and vermiculite (3:1 mass ratio) for 2 days. The roots were then drenched with the antagonistic bacterial suspension, 20mL per seedling. 24 hours after drenching, pre-cultured wheat grains containing *Fusarium oxysporum* (5g / plant) were inoculated around the roots. Three control groups were set up: control group 1 involved soaking the roots in sterile water and then inoculating with *Fusarium oxysporum*; control group 2 involved soaking the roots in the antagonistic bacterial suspension and then inoculating with sterile wheat grains; and control group 3 involved soaking the roots in sterile water and then inoculating with sterile wheat grains. On day 30, the disease incidence was investigated according to the classification and investigation methods for tobacco root and stem diseases in the National Standard of the People's Republic of China (GB / T23222-2008), and the disease index and control effect were calculated.

[0041] The disease index is calculated using the formula: Σ(disease level × number of plants at that disease level) / (maximum disease level × total number of plants) × 100%.

[0042] The formula for calculating the prevention and control effect is: (disease index of control group - disease index of treatment group) / disease index of control group × 100%.

[0043] Meanwhile, the effects of biocontrol bacteria on agronomic traits were determined according to the agronomic trait measurement methods in the Tobacco Industry Standard of the People's Republic of China (YC / T142-2010). The measurement indicators included the number of effective leaves of tobacco plants, maximum leaf length, maximum leaf width, aboveground fresh weight and root fresh weight. Leaf area (m²) was calculated as 0.6345 × leaf length (cm) × leaf width (cm).

[0044] The experimental results (Table 3) show that the biocontrol bacteria have a good control effect on Fusarium oxysporum root rot of tobacco. The incidence rate of the control treatment (CK) inoculated with Fusarium oxysporum was 100%, and the disease index was 81.00±7.67a. However, the treatment with biocontrol bacteria F3-31 can significantly reduce the incidence rate and disease index of Fusarium oxysporum root rot of tobacco, with an incidence rate of 0 and a disease index of 0.00±0.00b. The average control efficacy against Fusarium oxysporum root rot of tobacco is 100±0.00a.

[0045] Table 3: Control efficacy of biocontrol strains against Fusarium root rot in tobacco. The effect of promoting growth is as follows Figure 4As shown, the agronomic trait test results indicated that the number of effective leaves in the antagonistic bacteria-treated plants was not significantly different from that in the control group, but the maximum leaf area, aboveground fresh weight, root fresh weight, total root length, and total root surface area were all significantly improved compared to the control. The number of effective leaves in tobacco plants treated with antagonistic bacteria F3-31 increased by approximately 1.5 leaves.

[0046] Table 4: Effects of antagonistic bacteria on tobacco agronomic traits Example 6 Antagonistic bacteria F3-31 were preserved in glycerol tubes at -80℃ in our laboratory and activated on LB medium. Target pathogenic fungus: *Phytophthora tobaccoii* (… Phytophthora nicotianae ), Tobacco rhizosphere mold ( Thielaviopsis basicola ), Iris filamentosa ( Aphanomyces iridis All of these were isolated and preserved in our laboratory and activated on PDA medium. Among them, *Phytophthora nicotinea* caused tobacco black shank disease, *Rhizopus nicotinea* caused tobacco root black rot disease, and *Iris nicotinea* caused tobacco floating seedling root rot disease.

[0047] Take activated F3-31 and streak it parallel to both sides of a PDA plate (2.5 cm from the center) using a streak needle. Incubate at 28℃ for 24 h. Use a 5 mm punch to collect mycelial cakes from the edge of each of the three pathogenic fungi colonies and inoculate them into the center of the above plates. Use a plate without F3-31 as a control (CK). Incubate at 28℃ for 5-7 days (5 days for *Phytophthora indicum*, 7 days for the others). Measure the colony diameter of the pathogenic fungi in the control and treatment groups, and calculate the inhibition rate using the following formula:

[0048] Inhibition rate (%) = (Coronary diameter of blank control group - Colony diameter of confrontation experimental group) / (Coronary diameter of blank control group - Diameter of mycelial cake) × 100% Each treatment was repeated three times, and the results were averaged. The results showed that F3-31 exhibited significant antagonistic effects against all three pathogenic fungi, with inhibition rates ranging from 65% to 72% (Table 5). Among them, the inhibitory effect against Phytophthora tobaccois was the strongest, with an average inhibition rate of 71.5%; the inhibition rate against Mycotomyces irisis was 65.2%, both significantly higher than the control group (P<0.05).

[0049] Table 5: Antifungal effects of F3-31 against three pathogenic fungi in plate confrontation Example 7 The antibacterial activity of antagonistic bacterial fermentation filtrate against three pathogenic fungi was determined. Following the fermentation filtrate preparation and plate-drilling method described in Example 4: F3-31 was inoculated into LB liquid medium, incubated at 28°C with shaking at 180 rpm for 48 h, centrifuged at 12000 rpm for 10 min, and the supernatant was sterilized through a 0.22 μm filter membrane to obtain sterile fermentation filtrate. Two 9 mm diameter wells were symmetrically drilled 2.5 cm from the center of a PDA plate, and 200 μL of fermentation filtrate was injected into each well, with sterile LB liquid medium as a control. After the filtrate was absorbed, a 5 mm diameter pathogenic fungal mycelium was inoculated into the center of the plate, incubated at 28°C for 5-7 days, and the colony diameter was measured. The inhibition rate was calculated (formula same as in Example 6). Each treatment was repeated three times.

[0050] The results showed that the F3-31 fermentation filtrate had an inhibitory effect on the mycelial growth of the three pathogenic fungi that was similar to that on the live bacteria, with an inhibition rate between 64% and 70% (Table 6), indicating that its antibacterial activity may be related to the metabolites produced during fermentation.

[0051] Table 6: Antibacterial effect of F3-31 fermentation filtrate against three pathogenic fungi Example 8 Following the pot experiment method in Example 5, tobacco variety K326 was used as the test crop. F3-31 cells were cultured to the logarithmic growth phase, centrifuged to collect the cells, and the concentration was adjusted to 10 with sterile physiological saline. 8 A bacterial suspension was prepared at CFU / mL. Tobacco seedlings (large cross stage) were soaked in the bacterial suspension for 30 min before transplanting. Two days later, each seedling was drenched with 20 mL of the bacterial suspension. 24 h later, the rhizosphere was inoculated with the pathogenic fungus (Phytophthora tobaccois spore suspension 1×10⁻⁶). 6 The concentration of *Rhizoctonia solani* was 20 mL per plant; both *Rhizoctonia solani* and *Iris spp.* were inoculated with infected wheat grains (5 g per plant). Control groups were divided into three groups: CK1: tobacco seedlings treated with sterile water and then inoculated with the pathogenic fungus; CK2: tobacco seedlings treated with F3-31 bacterial suspension and then inoculated with sterile wheat grains / sterile water; CK3: tobacco seedlings treated with sterile water and then inoculated with sterile wheat grains / sterile water. After 30 days of culture, the disease index was investigated according to GB / T23222-2008, and the control effect was calculated.

[0052] Disease index = Σ (disease level × number of plants at that disease level) / (maximum disease level × total number of plants) × 100.

[0053] Prevention and control effect (%) = (disease index of control group - disease index of treatment group) / disease index of control group × 100.

[0054] The results showed that the F3-31 treatment was significantly effective against diseases caused by the three pathogenic fungi, with a control efficacy of 92.6% against Phytophthora indicum and 89.5% and 85.3% against Root Flocculation and Iris Fibromycosis, respectively (Table 7).

[0055] Table 7: Potted plant control efficacy of F3-31 against three pathogenic fungal diseases This invention has screened out a species of Fusarium oxysporum with good antagonistic effects, exhibiting strong inhibitory effects against various common tobacco pathogenic fungi, significantly inhibiting the mycelial growth of the target pathogen Fusarium oxysporum, and showing good potted plant control efficacy against Fusarium root rot. The application of this invention in the control of Fusarium root rot in tobacco makes it have broad market prospects.

[0056] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0057] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0058] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A type of *Cathaya argyrophylla*, characterized in that, The *Cinnamomum cassia* species is identified as F3-31, and its classification name is... Kitasatospora cathayae It was deposited on December 18, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.33119.

2. A microbial agent, characterized in that, It is prepared from the *Cathaya argyrophylla* strain described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The microbial agent is a suspension or fermentation broth.

4. The application of the *Cathaya argyrophylla* fungus as described in claim 1 or the fungal agent as described in any one of claims 2 to 3 in the prevention and control of tobacco root rot, tobacco black shank disease, tobacco root black rot, and tobacco floating seedling root rot.

5. The application according to claim 4, characterized in that, The tobacco root rot mentioned is Fusarium root rot of tobacco.

6. The application according to claim 5, characterized in that, Before planting, the roots of tobacco seedlings are soaked in a suspension of *Cathaya argyrophylla* to prevent Fusarium root rot in tobacco.

7. The application according to claim 5, characterized in that, After planting, the tobacco seedlings were treated with a suspension of the aforementioned *Cathaya argyrophylla* fungus by root irrigation.

8. The application of *Cathaya argyrophylla* sp. as described in claim 1 in the control of pathogenic fungi, characterized in that, The pathogenic fungi include Fusarium oxysporum, Phytophthora nicotineum, Rhizoctonia solani, and Mycotomyces iris.

9. The application of *Cryptostrobus tinctoria* as described in claim 1 in improving tobacco agronomic traits, characterized in that... The agronomic traits of tobacco are leaf area, aboveground fresh weight, root fresh weight, root length, root surface area, and root volume.

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