Application of LtEgID9 gene in regulating the pathogenicity and stress resistance of Lasiodiplodia theobromae
By mining and regulating the LtEgID9 gene, a transgenic strain of Lasiodiplodia theobromae was prepared using transformation methods, which solved the problem of the weak pathogenic mechanism of the pathogen and realized a new approach to disease control.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- GUIZHOU UNIV
- Filing Date
- 2026-02-13
- Publication Date
- 2026-05-29
AI Technical Summary
Current technologies have limited molecular biological research on the pathogen Lasiodiplodia theobromae, and there is a lack of reports on its pathogenic mechanisms, making it difficult to develop effective green control strategies.
By mining the LtEgID9 gene and regulating the pathogenicity and stress resistance of Lasiodiplodia theobromae, transgenic strains were prepared by knocking down or overexpressing LtEgID9 protein using PEG-mediated protoplast transformation to reduce its pathogenicity and stress resistance.
Without affecting the growth of Lasiodiplodia theobromae, the pathogenicity and stress resistance of the disease were significantly reduced, providing new targets for disease control and drug development.
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Figure CN122104762A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to the application of the LtEgID9 gene in regulating the pathogenicity and stress resistance of Lasiodiplodiatheobromae. Background Technology
[0002] Tea (Camellia sinensis (L.) O. Kuntze) is a perennial evergreen shrub or small tree, used as an important economic crop. Our research group has isolated and identified several pathogenic fungi from tea leaves in various regions, including *Botrytis cinerea*, *L. theobromae*, *Didymella segeticola*, and *Epicoccum sorghinum*, which can cause diseases in tea leaves. These pathogens seriously affect the yield and quality of tea leaves. Previously, fungicides and other chemical pesticides were mainly used to control fungal diseases in crops. However, safety concerns such as pesticide residues, damage to non-target organisms, and increased fungal resistance have led the scientific community to pay increasing attention to the use of pesticides. Therefore, in-depth analysis of the pathogenic molecular mechanisms of these fungi and the identification of key pathogenic factors are prerequisites for developing green and efficient control strategies.
[0003] To date, molecular biological research on *L. theobromae* pathogens, both domestically and internationally, remains relatively weak, particularly lacking reports on the pathogenic mechanisms. Therefore, fully exploring *L. theobromae* pathogenicity-related genes and conducting functional studies will contribute to a comprehensive understanding of the pathogenic mechanisms of *L. theobromae* and the control of the diseases it causes. This invention obtains the LtEgID9 gene from *L. theobromae*, which is associated with the pathogenicity and stress resistance of *L. theobromae*. Therefore, the LtEgID9 gene will have significant importance and application prospects in the control of diseases caused by *L. theobromae* and the breeding of new disease-resistant plant varieties. Summary of the Invention
[0004] In view of this, one of the objects of the present invention is to provide a new use for the LtEgID9 protein derived from L. theobromae, namely, the use of the LtEgID9 protein in regulating the pathogenicity and / or stress resistance of L. theobromae, wherein the LtEgID9 protein is a protein with the amino acid sequence shown in SEQ ID NO.2 or a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein with the sequence shown in SEQ ID NO.2.
[0005] A second objective of this invention is to provide the application of biomaterials related to the LtEgID9 protein in regulating the pathogenicity and / or stress resistance of L. theobromae. The biomaterials include nucleic acid molecules encoding the LtEgID9 protein or expression cassettes containing the nucleic acid molecules, recombinant vectors, or recombinant microorganisms. For example, the biomaterials may also be recombinant vectors or recombinant microorganisms containing the expression cassettes.
[0006] Preferably, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO.1. The nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA, or RNA, such as mRNA or hnRNA. The vector can be a plasmid, granule, bacteriophage, or viral vector; the microorganism can be yeast, bacteria, algae, or fungi, such as Agrobacterium.
[0007] A third objective of this invention is to provide the application of the LtEgID9 protein as a target in the design and screening of antifungal drugs.
[0008] The fourth objective of this invention is to provide the application of the above-mentioned LtEgID9 protein or the above-mentioned biological material in the cultivation of transgenic L. theobromae with reduced pathogenicity and / or reduced resistance to stress.
[0009] A fifth objective of this invention is to provide a method for cultivating transgenic *L. theobromae* with reduced pathogenicity and / or reduced resistance to adverse conditions, comprising the step of reducing the expression level and / or activity of the aforementioned LtEgID9 protein in the receptor *L. theobromae* to obtain transgenic *L. theobromae* (such as an LtEgID9 knockdown strain). The pathogenicity and / or resistance to adverse conditions of the transgenic *L. theobromae* are lower than those of the wild-type *L. theobromae*.
[0010] Preferably, the method for reducing the expression level and / or activity of LtEgID9 protein in the receptor L. theobromae is achieved by inhibiting, knocking out, or silencing the expression of the gene encoding LtEgID9 protein in the receptor L. theobromae.
[0011] Preferably, the inhibition method is to use PEG-mediated protoplast transformation to knock down the gene encoding the LtEgID9 protein in the receptor L. theobromae.
[0012] Preferably, the PEG-mediated protoplast transformation method involves introducing a knockdown vector into the protoplasts of the receptor L. theobromae.
[0013] The sixth objective of this invention is to provide the application of the above-described method in the prevention and control of diseases caused by L. theobromae.
[0014] This invention provides the application of the LtEgID9 protein and its encoding gene derived from *L. theobromae* in regulating the pathogenicity and stress resistance of the plant pathogenic fungus *L. theobromae*. Knockdown of the LtEgID9 protein encoding gene in wild-type *L. theobromae* significantly reduced both pathogenicity and stress resistance without affecting its growth. Therefore, LtEgID9 can serve as a target for fungicides and a key protein in the pathogenic mechanism of diseases, enabling the development of agents for diseases caused by *L. theobromae* infection and the breeding of new resistant varieties, showing broad application prospects in the control of plant pathogenic fungal diseases. Attached Figure Description
[0015] Figure 1 A diagram of the protoplasts prepared according to this invention;
[0016] Figure 2 This is a diagram showing the colony morphology and growth diameter of wild-type L. theobromae, RNAi-LtEgID9, and OE-LtEgID9 on PDA medium according to the present invention.
[0017] Figure 3 The figures show the growth of L. theobromae wild-type, OE-LtEgID9 and RNAi-LtEgID9 under different stress conditions.
[0018] Figure 4 This is a graph showing the relative growth inhibition rates of L. theobromae wild-type, OE-LtEgID9, and RNAi-LtEgID9 under different stress conditions.
[0019] Figure 5 This invention represents the relative expression levels of LtEgID9 at different time stages in tea infected with wild-type L. theobromae.
[0020] Figure 6 The relative expression levels of LtEgID9 in wild-type L. theobromae, RNAi-LtEgID9, and OE-LtEgID9 after 36 h of growth on PDA plates are shown in the figure.
[0021] Figure 7 The graph shows the results of pathogenicity assays of wild-type L. theobromae, RNAi-LtEgID9, and OE-LtEgID9 on tea according to this invention. Detailed Implementation
[0022] The present invention will be described in detail below with reference to embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of application of the present invention. The present invention is not limited to the following embodiments or examples. Any modifications and variations made without departing from the spirit of the present invention should be included within the scope of the present invention. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available.
[0023] 1. Data: The CDS sequence of the LtEgID9 gene is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2.
[0024] 2. The strain used in this embodiment is L. theobromae, strain number: GZHS-2017-010 (accession number: CGMCC3.20151). All of the above strains were isolated and obtained by the inventors' team and can be obtained from the National Key Laboratory of Green Pesticides of Guizhou University.
[0025] Example 1: LtEgID9 gene knockdown of L. theobromae
[0026] 1. A 248 bp fragment from the LtEgID9 gene was amplified by PCR using specific primers, as shown in Table 1. The PCR program was set to 94 ℃ pre-deformation for 2 min, 98 ℃ denaturation for 10 s, 55 ℃ annealing for 30 s, 72 ℃ extension for 50 s, for 35 cycles, followed by a final extension at 72 ℃ for 5 min. After PCR, 50 μL was collected for gel purification. The gel purification was performed using the Trelief DNAGel Extraction Kit (Beijing Qingke Biotechnology). For specific purification steps, please refer to the instruction manual.
[0027] Table 1 Primer fragments for target gene amplification
[0028]
[0029] Fragments in both the reverse and forward directions were amplified using primers with adapter sequences. EgID9-Ri-PF1 introduced a Hind III restriction site sequence at the 5' end of EgID9-Ri-R, and EgID9-Ri-SR1 introduced an EcoRI restriction site sequence at the 3' end of EgID9-Ri-F. The primers were modified according to primer design principles, as shown in Table 2. The amplified and recovered fragments were cloned and ligated into the pMD18-T vector (TaKaRa) to form a double-stranded DNA fragment with a hairpin structure. The constructed hairpin fragment was then digested with Hind III and EcoRI and inserted into the corresponding sites after digestion in the pBluescriptII KS vector (carrying the G418 resistance gene) to construct the RNAi expression vector.
[0030] Table 2 Primers for RNAi body construction
[0031]
[0032] 2. Preparation of L. theobromae protoplasts
[0033] 1) Inoculate L. theobromae into potato dextrose broth (PDB) medium and culture at 25°C for 36 h. Collect the mycelia into 2 mL centrifuge tubes, break the mycelia with a grinder, transfer the mycelial suspension to CM liquid medium and shake for 36 h. Filter and collect the fresh mycelia of L. theobromae.
[0034] 2) Using 10 mL of 0.7 mol / L sodium chloride solution as an osmotic pressure stabilizer, a mixed enzymatic hydrolysate of drislase and snailase was prepared. 5 g of suspended mycelium was lysed and enzymatically hydrolyzed at 28℃ and 100 rpm for 4 h.
[0035] 3) Filter through 2-3 layers of sterile lens paper (fiber mesh size 45±12μm), wash with 0.7 mol / L sodium chloride, collect the filtrate, and centrifuge at 4000 rpm for 6 min at 4℃. Resuspend in 15 mL of 1.2 mol / L sorbitol buffer (STC) solution;
[0036] 4) Discard the supernatant and resuspend the protoplast pellet in 1 mL of STC buffer, as follows: Figure 1 As shown, a concentration of 1×10⁻⁶ was prepared. 7 Prepare a protoplast suspension of 1 protoplast per mL and place it on ice until needed.
[0037] 3. L. theobromae protoplast transformation
[0038] 1) Take 200 μL of protoplast suspension and add it to a 50 mL centrifuge tube. Add 10–20 μg of RNAi expression vector, mix gently, and incubate on ice for 20 min.
[0039] 2) Add 1.2 mL of 40% polyethylene glycol 3350 buffer (PTC) in three portions, gently invert and mix, let stand at room temperature for 20 min, add 5 mL of TB3 liquid culture medium, invert and mix, and incubate at 25℃ and 120 rpm for 12–16 h.
[0040] 3) Centrifuge at 4000 rpm / min for 6 min at room temperature, discard the supernatant, and resuspend the remaining 1 mL of resuspension with the regenerated protoplasm.
[0041] 4) Add to 50 mL of warm TB3 regeneration solid medium, mix well and prepare plates. Incubate upside down at 25℃ for 24 h, then cover with PDA medium containing 30 μg / mL G418 and incubate at 25℃ for about 2-4 days until transformants grow. Then subculture and screen for 3 generations.
[0042] 4. Transformer Validation
[0043] After three generations of subculture on PDA medium containing G418, the corresponding G418 positive transformants were screened to obtain the LtEgID9 knockdown L. theobromae strain (RNAi-LtEgID9).
[0044] Example 2: Overexpression of the LtEgID9 gene in L. theobromae
[0045] 1. The LtEgID9 gene was amplified by PCR using specific primers, as shown in Table 3. The PCR program was set to 94℃ pre-deformation for 2 min, 98℃ denaturation for 10 s, 55℃ annealing for 30 s, 72℃ extension for 50 s, for 35 cycles, followed by a final extension at 72℃ for 5 min. After PCR, 50 μL was collected for gel purification. The gel purification was performed using the Trelief DNA Gel Extraction Kit (Beijing Qingke Biotechnology). For specific purification steps, please refer to the instruction manual.
[0046] Table 3 LtEgID9 gene amplification primer fragments
[0047]
[0048] The pBluescript II KS vector was digested with Hind III and Pst I restriction enzymes, and primers containing homologous arms of the pBluescript II KS vector were designed. The primer sequences are shown in Table 4. The purified LtEgID9 gene was amplified using the primers, and the target sequence was ligated into the pBluescript II KS vector through homologous recombination to obtain the overexpression vector.
[0049] Table 4 Primers for overexpression vector construction
[0050]
[0051] The preparation, transformation, and verification of L. theobromae protoplasts were the same as in Example 1.
[0052] Results: The overexpression vector was transformed into L. theobromae protoplasts, and the corresponding G418 positive transformants were obtained by screening, which resulted in the LtEgID9 overexpressing L. theobromae strain (OE-LtEgID9).
[0053] Example 3: Phenotypic observation and stress resistance analysis of wild-type L. theobromae, OE-LtEgID9, and RNAi-LtEgID9.
[0054] 1. Colony morphology observation and growth rate determination
[0055] Wild-type L. theobromae, RNAi-LtEgID9, and OE-LtEgID9 were inoculated onto PDA medium and cultured at 25°C in the dark for 36 h. The colony diameter was then measured using the cross-crossing method, and the colony morphology was observed. Each treatment was replicated in triplicate.
[0056] The colony morphology and growth diameter of *L. theobromae* wild-type, RNAi-LtEgID9, and OE-LtEgID9 on PDA medium are as follows: Figure 2 As shown, where, Figure 2 A shows an observation of colony morphology; Figure 2 B is a graph showing colony diameter, with the vertical axis representing colony diameter. Values are the averages (mean ± SD) of three independent experiments. One-way ANOVA was used to determine statistical significance; different lowercase letters indicate significant differences between groups (P < 0.05). Scale bar = 1 cm. Figure 2It can be seen that the colony morphology and growth diameter of wild-type L. theobromae, RNAi-LtEgID9 and OE-LtEgID9 on PDA medium are similar, indicating that the LtEgID9 gene does not affect the growth of L. theobromae.
[0057] 2. Analysis of stress resistance
[0058] 1) Analysis of high osmotic pressure stress
[0059] Wild-type L. theobromae, OE-LtEgID9, and RNAi-LtEgID9 were inoculated onto PDA medium containing 0.5 mol / L sorbitol and 0.6 mol / L NaCl, respectively, and incubated upside down at 25°C for 36 h. The colony growth of wild-type L. theobromae, OE-LtEgID9, and RNAi-LtEgID9 strains was then observed.
[0060] Measure the colony diameter of all strains (using the cross-hatching method) and record the data by photograph. Calculate the growth inhibition rate of the strains = (colon diameter of control strain - colony diameter of treated strain) / colony diameter of control strain × 100%.
[0061] 2) Oxidative stress analysis: Wild-type L. theobromae, OE-LtEgID9 and RNAi-LtEgID9 were inoculated on PDA medium containing 0.06% H2O2 (V:V) and incubated upside down in an incubator at 25℃ for 36 h. The colony growth of wild-type L. theobromae, OE-LtEgID9 and RNAi-LtEgID9 was then observed.
[0062] 3) Cell wall integrity analysis
[0063] Wild-type L. theobromae, OE-LtEgID9, and RNAi-LtEgID9 were inoculated onto PDA medium containing 120 μg / mL Congo red and incubated upside down at 25°C for 36 h. The colony growth of wild-type L. theobromae, OE-LtEgID9, and RNAi-LtEgID9 strains was then observed.
[0064] 4) Cell membrane integrity analysis
[0065] Wild-type L. theobromae, OE-LtEgID9, and RNAi-LtEgID9 were inoculated onto PDA medium containing 0.05% sodium dodecyl sulfate (SDS) (V:V) and incubated upside down at 25°C for 36 h. The colony growth of wild-type L. theobromae, OE-LtEgID9, and RNAi-LtEgID9 strains was then observed.
[0066] The growth of L. theobromae wild-type, OE-LtEgID9, and RNAi-LtEgID9 under different stress conditions is as follows: Figure 3 As shown (where, Figure 3 The first row shows the growth of wild-type L. theobromae strains on culture media under different stress conditions; Figure 3 The second row shows the growth of OE-LtEgID9 on culture media under different stress conditions; Figure 3 The third row shows the growth of RNAi-LtEgID9 on different stress conditions; 120 μg / mL Congo Red: containing a final concentration of 120 μg / mL Congo Red; 0.5 mol / L Sorbitol: containing a final concentration of 0.5 mol / L Sorbitol; 0.05% SDS: containing a final concentration of 0.05% SDS (V:V); 0.06% H2O2: containing a final concentration of 0.06% H2O2 (V:V); 0.6 mol / L NaCl: containing a final concentration of 0.6 mol / L NaCl); the relative growth inhibition rates of L. theobromae wild-type, OE-LtEgID9, and RNAi-LtEgID9 under different stress conditions are shown in the figure. Figure 4 As shown, the vertical axis represents the relative growth inhibition rate, and the values are based on the mean (±SE) of three independent experiments. One-way ANOVA was used to determine statistical significance. * indicates P<0.05, ** indicates P<0.01, and ns indicates no significant difference between groups. Figure 4 From left to right, the figures show the relative growth inhibition rates of *L. theobromae* wild-type, OE-LtEgID9, and RNAi-LtEgID9 colonies under stress from Congo red, sorbitol, SDS, H2O2, and NaCl. Figure 3 and Figure 4The results showed that in medium containing 0.06% H2O2, the relative growth inhibition rate of RNAi-LtEgID9 colonies was significantly higher than that of wild-type, while the relative growth inhibition rate of OE-LtEgID9 colonies was significantly lower than that of wild-type. This indicates that the LtEgID9 gene can affect the sensitivity of *L. theobromae* to H2O2, and that knockdown of the LtEgID9 gene increases sensitivity to H2O2. In conclusion, knockdown of the LtEgID9 gene significantly reduces tolerance to oxidative stress.
[0067] Example 3: Detection of relative expression levels of LtEgID9 in wild-type L. theobromae, RNAi-LtEgID9, and OE-LtEgID9, and analysis of pathogenicity.
[0068] The relative expression level of the LtEgID9 gene was determined by extracting RNA from *L. theobromae* using the TransZol Up high-performance RNA extraction kit (Beijing TransGen Biotech), and then reverse transcribing it into single-stranded cDNA using a reverse transcription kit. The fragment was amplified using primer pairs EgID9-qPCR-F / EgID9-qPCR-R (Table 5). The relative expression level of the target gene was detected by RT-qPCR. One-way ANOVA was used to determine statistical significance; different lowercase letters represent significant differences between groups (P < 0.05). The highest relative expression level of LtEgID9 was found at 12 hours. Figure 5 Therefore, the relative expression levels of LtEgID9 in wild-type L. theobromae, RNAi-LtEgID9, and OE-LtEgID9 were detected at 12 hours, and one-way ANOVA was used to determine statistical significance. *** indicates P < 0.001, **** indicates P < 0.0001.
[0069] Wild-type *L. theobromae*, RNAi-LtEgID9, and OE-LtEgID9 were inoculated onto PDA medium and incubated upside down at 25°C for 24 h. Several mycelial cakes were then collected from the edge of the colonies using a 4 mm diameter sterile punch and inoculated onto the surface of *Camellia sinensis* cv. Fuding-dabaicha leaves. The size of lesions caused by wild-type *L. theobromae* infection at different time points was measured. Two days after infection, the disease incidence of the three strains on tea was investigated. One-way ANOVA was used to determine statistical significance. ** indicates P < 0.01, **** indicates P < 0.0001. Scale bar = 1 cm.
[0070] Table 5 Primers for overexpression and RNAi transformants-RT-qPCR detection
[0071]
[0072] The relative expression levels of LtEgID9 in L. theobromae wild-type, RNAi-LtEgID9, and OE-LtEgID9 were detected after 36 hours of growth on PDA plates as follows: Figure 6 As shown, the relative expression level of LtEgID9 was significantly lower in RNAi-LtEgID9 compared to wild-type L. theobromae, while the relative expression level of OE-LtEgID9 was significantly higher.
[0073] The results of pathogenicity assays for wild-type L. theobromae, RNAi-LtEgID9, and OE-LtEgID9 in tea are as follows: Figure 7 As shown, where, Figure 7 A shows the lesions on tea leaves 2 days after inoculation with wild-type L. theobromae, RNAi-LtEgID9, and OE-LtEgID9. Figure 7 Figure B shows the lesion area measurements of *L. theobromae* wild-type, RNAi-LtEgID9, and OE-LtEgID9 inoculated on tea leaves 2 days after inoculation. The vertical axis represents lesion length. One-way ANOVA was used to determine statistical significance. ** indicates P < 0.01, **** indicates P < 0.0001. Scale bar = 1 cm. Figure 7 It can be seen that, compared with wild-type L. theobromae, the lesion length of OE-LtEgID9 is significantly increased in tea leaves, indicating that the LtEgID9 gene has a significant impact on the pathogenicity of L. theobromae. On the other hand, the lesion length of RNAi-LtEgID9 is significantly decreased, indicating that the pathogenicity of L. theobromae is significantly inhibited after knocking down the LtEgID9 gene.
[0074] In summary, the LtEgID9 gene can regulate the pathogenicity of *L. theobromae*. The LtEgID9 gene provided by this invention can be used to control diseases caused by *L. theobromae* without affecting its growth, and can serve as a target for drugs used in plant disease control. Those skilled in the art can follow this specification to develop fungicides for the control of plant diseases, especially those caused by *L. theobromae*.
[0075] The conventional techniques and solutions not described in detail in the above embodiments are all well known in the art, and therefore will not be elaborated upon here. The above embodiments and / or experimental examples describe the preferred embodiments of the present invention in detail. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within the protection scope of the present invention.
Claims
1. The application of LtEgID9 protein in regulating the pathogenicity and / or resilience of Lasiodiplodia theobromae, characterized in that, The LtEgID9 protein is a protein with the amino acid sequence shown in SEQ ID NO.2 or a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of a protein with the sequence shown in SEQ ID NO.
2.
2. The application of the LtEgID9 protein expression-related biomaterial as described in claim 1 in regulating the pathogenicity and / or stress resistance of L. theobromae, characterized in that, The biological material includes a nucleic acid molecule encoding the LtEgID9 protein or an expression cassette, recombinant vector or recombinant microorganism containing the nucleic acid molecule.
3. The application as described in claim 2, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.
1.
4. The use of the LtEgID9 protein as a target in the design and screening of anti-L. theobromae drugs, as described in claim 1, is characterized in that... The anti-L. theobromae drug reduces the expression of LtEgID9 protein.
5. The use of the LtEgID9 protein as described in claim 1 or the biological material as described in claim 2 or 3 in the cultivation of transgenic L. theobromae with reduced pathogenicity and / or reduced resistance to stress.
6. A method for breeding transgenic *L. theobromae* with reduced pathogenicity and / or reduced resistance to adverse conditions, characterized in that, The procedure includes the step of reducing the expression level and / or activity of the LtEgID9 protein as described in claim 1 in the receptor L. theobromae to obtain transgenic L. theobromae.
7. The method as described in claim 6, characterized in that, The method for reducing the expression level and / or activity of the LtEgID9 protein as described in claim 1 in the receptor L. theobromae is achieved by inhibiting, knocking out, or silencing the gene encoding the LtEgID9 protein in the receptor L. theobromae.
8. The method as described in claim 7, characterized in that, The inhibition method is a PEG-mediated protoplast transformation method.
9. The method as described in claim 8, characterized in that, The PEG-mediated protoplast transformation method involves introducing a knockdown vector into the protoplasts of the receptor L. theobromae.
10. The application of the method according to any one of claims 6-9 in the prevention and control of diseases caused by L. theobromae.