Trichoderma guepinii gudj16 and application thereof
By screening Trichoderma rhododendron strain GUDJ16 and its spore suspension, the problem of controlling soft rot in kiwifruit was solved, achieving highly efficient inhibition of *Trichoderma variegata* and significant reduction of soft rot in kiwifruit.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- GUIZHOU UNIV
- Filing Date
- 2025-12-23
- Publication Date
- 2026-05-29
AI Technical Summary
Kiwi soft rot is caused by pathogenic fungi such as *Botrytis cinerea*, and current technologies are insufficient to effectively control it, thus affecting fruit safety.
The *Trichoderma rhododendron* strain GUDJ16 was isolated and screened, and its spore suspension and microbial agent were prepared and applied to the surface treatment of kiwifruit to prevent soft rot.
Trichoderma rhododendron GUDJ16 showed a 77.15% inhibition rate against grape phytoestrogens and a 61.26% control effect against soft rot in kiwifruit, demonstrating good control efficacy.
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Figure CN121450442B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial technology, and in particular to a Trichoderma rhododendron GUDJ16 and its applications. Background Technology
[0002] Kiwifruit soft rot is a disease caused by pathogenic fungi, seriously threatening the safety of kiwifruit during ripening and post-harvest storage. Studies have shown that *Botryosphaeria dothidea* is one of the main pathogens. Infected kiwifruit exhibit symptoms such as collapsed skin, softened tissue, and a rotten odor. After peeling away the epidermis at the lesion site, a milky-white area is revealed in the center, surrounded by a yellowish-green transition zone. The flesh tissue shows signs of softening and rotting. Longitudinal sectioning reveals that the soft rot area extends in a cone shape into the flesh, making it highly susceptible to spread.
[0003] Trichoderma spp. are widely found in soil, plant rhizosphere and other natural environments. They have good antagonistic effects against a variety of plant pathogens and are an important representative in the research of biocontrol fungi. Summary of the Invention
[0004] The purpose of this invention is to provide a Trichoderma rhombifolia strain, GUDJ16, and its applications to solve the problems existing in the prior art. This invention isolates and screens a Trichoderma rhombifolia strain, GUDJ16, which exhibits good control effects against soft rot in kiwifruit and has high application value in the field of kiwifruit soft rot control.
[0005] To achieve the above objectives, the present invention provides the following solution:
[0006] In the first aspect, the present invention provides a Trichoderma rhododendron GUDJ16, which was deposited at the China Center for Type Culture Collection (CCTCC) on September 11, 2025, at Wuhan University, Wuhan, China, with accession number CCTCC NO:M 20252004.
[0007] Secondly, the present invention also provides a spore suspension containing spores of the aforementioned Trichoderma rhododendron GUDJ16.
[0008] Thirdly, the present invention also provides a method for preparing the spore suspension, comprising the steps of inoculating the Trichoderma rhododendron GUDJ16 into a culture medium and culturing it for 5 days, and then washing the culture to obtain the spore suspension.
[0009] Fourthly, the present invention also provides the application of the aforementioned Trichoderma rhododendron GUDJ16 or the aforementioned spore suspension in the preparation of microbial inoculants.
[0010] Fifthly, the present invention also provides a microbial inoculant, wherein the microbial inoculant includes the aforementioned Trichoderma rhododendron GUDJ16 or the aforementioned spore suspension.
[0011] In a sixth aspect, the present invention also provides the application of the aforementioned Trichoderma rhododendron GUDJ16, the aforementioned spore suspension, or the aforementioned microbial agent in the control of plant pathogens.
[0012] Preferably, the plant pathogen is selected from one or more of the following: *Botrytis cinerea*, *Fusarium solani*, *Fusarium graminearum*, *Fusarium asiaticum*, *Rhizoctonia solani*, *Sclerotium sclerotiorum*, *Anthracnose fungus of banana*, *Trichophyton mentagrophytes*, and *Alternaria spp.*
[0013] In a seventh aspect, the present invention also provides the application of the aforementioned Trichoderma rhododendron GUDJ16, the aforementioned spore suspension, or the aforementioned microbial agent in the preparation of products for the prevention and control of plant pathogens.
[0014] In an eighth aspect, the present invention also provides a product for preventing and controlling plant pathogens, the product comprising the aforementioned Trichoderma rhododendron GUDJ16, the aforementioned spore suspension, or the aforementioned microbial agent;
[0015] The plant pathogens are selected from one or more of the following: Staphylococcus aureus, Fusarium solani, Fusarium graminearum, Fusarium asiaticum, Rhizoctonia solani, Sclerotium sclerotiorum, Banana anthracnose fungus, Bean intercropping scabies, and Alternaria spp.
[0016] In a ninth aspect, the present invention also provides a method for preventing and controlling plant pathogens, the method comprising the step of applying the Trichoderma rhododendron GUDJ16, the spore suspension, or the microbial agent to the plant;
[0017] The plant pathogens are selected from one or more of the following: Staphylococcus aureus, Fusarium solani, Fusarium graminearum, Fusarium asiaticum, Rhizoctonia solani, Sclerotium sclerotiorum, Banana anthracnose fungus, Bean intercropping scabies, and Alternaria spp.
[0018] The present invention discloses the following technical effects:
[0019] This invention isolated and screened a Trichoderma rhombifolia strain, GUDJ16, which exhibits good control effects against soft rot in kiwifruit. It was found that its inhibition rate against *Botryosphaeria dothidea* reached 77.15%, while also showing inhibitory effects against *Fusarium solani*, *Fusarium graminearum*, *Fusarium oxysporum*, *Rhizoctonia solani*, *Sclerotium sclerotium*, *Anthracnose fungus*, *Trichoderma bean*, and *Alternaria alternata*. Verification of its control effect on detached fruits infected with soft rot in kiwifruit showed that treatment with GUDJ16 achieved a control effect of 61.26% against soft rot, demonstrating that GUDJ16 has high application value in the control of soft rot in kiwifruit.
[0020] Preservation information: Trichoderma rhododendron GUDJ16 was deposited at the China Center for Type Culture Collection (CCTCC) on September 11, 2025, at Wuhan University, Wuhan, China, with accession number CCTCC NO:M20252004. Attached Figure Description
[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0022] Figure 1 The image shows the antibacterial effect of Trichoderma rhodopseudomonas GUDJ16, screened in this invention, against Botrytis cinerea; where A is a plate image of B. dothidea growing alone, and B is a plate image of colonies co-cultured with B. dothidea.
[0023] Figure 2 The morphological characteristics of Trichoderma rhodops GUDJ16 screened in this invention are shown below; where A is a growth image of colonies on PDA medium, B is a growth image of colonies on SNA medium, C is a growth image of colonies on CMA medium, D is a conidia of colonies, and EI is a conidiophore and phialid of colonies; the scale bar of Figure D to Figure I is 10 μm.
[0024] Figure 3 A phylogenetic tree was constructed using the maximum likelihood method based on the sequences of multiple genes (rpb2 and tef1).
[0025] Figure 4The study aimed to investigate the control effect of Trichoderma rhododendron GUDJ16, screened in this invention, on detached fruits infected with soft rot of kiwifruit; AC was the negative control group, DF was the positive control group, and GI was the Trichoderma rhododendron GUDJ16 treatment group.
[0026] Figure 5 The inhibitory effect of volatile substances from *Trichoderma rhododendron* GUDJ16 on *B. dothidea* was investigated; AB was the control group, and CD was the treatment group.
[0027] Figure 6 The antibacterial effect and inhibition rate of Trichoderma rhodopseudomonas GUDJ16 against eight pathogens were investigated. Detailed Implementation
[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0029] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.
[0030] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.
[0031] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.
[0032] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.
[0033] Example 1
[0034] 1. Experimental Materials
[0035] 1.1 Soil Sample
[0036] Rhizosphere soil of Rhododendron axillaryum was collected from the Wumeng Grassland in Liupanshui City, Guizhou Province (104°37′E, 26°11′N).
[0037] 1.2 Target pathogens
[0038] The pathogen of kiwifruit soft rot (Botryosphaeria dothidea) was provided by the Tobacco College of Guizhou University, Guiyang City, Guizhou Province.
[0039] 1.3 Test Culture Medium
[0040] PDA medium: 200 g peeled potatoes, 20 g glucose, 15-20 g agar powder, 1 L sterile distilled water, natural pH, sterilized at 121℃ for 20 min before use.
[0041] Low-nutrient agar medium (SNA): KH2PO4 1.0 g, KNO3 1.0 g, MgSO4·7H2O 0.5 g, KCl 0.5 g, glucose 0.2 g, sucrose 0.2 g, agar 12.0 g, distilled water to a final volume of 1.0 L, sterilize at 121℃ for 20 min before use.
[0042] Corn flour agar medium (CMA): 7.0 g corn extract, 15.0 g agar, pH 6.0 ± 0.2, distilled water to a final volume of 1.0 L, sterilize at 121℃ for 20 min before use.
[0043] 2. Experimental Methods
[0044] 2.1 Isolation, purification and preservation of strains
[0045] Forty-one strains were isolated and purified from the collected soil using the soil dilution plate coating method (a conventional method, which will not be described in detail here). These strains were then placed in cryovials containing 15-20% glycerol and stored at 4°C.
[0046] 2.2 Screening of biocontrol strains
[0047] Using *Botryosphaeria dothidea* as an indicator bacterium, the plate confrontation method was employed to screen biocontrol bacteria. The isolated and screened strains and indicator pathogen mycelia were inoculated separately at 2 cm margins on PDA medium, with an indicator pathogen mycelia serving as a control. Each treatment was performed in triplicate. The medium was incubated in the dark at 28°C, and the growth of the strains was observed and photographed periodically.
[0048] The formula for calculating the antibacterial rate is: Antibacterial rate = [(CT) / C] × 100%.
[0049] Where C and T represent the average growth radius of the tested pathogens in the control group and treatment group, respectively.
[0050] 2.3 Identification of biocontrol strains
[0051] 2.3.1 Morphological observation
[0052] The selected strains were inoculated into PDA medium and cultured in the dark at 28°C. During this period, the growth performance of the strains on the medium was observed, including colony color and sporulation status. Subsequently, the morphological characteristics of conidiophores, conidia (n=50), and sporulation structures were observed at 40x magnification (1×40) using a Zeiss microscope (Carl ZeissMicroscopy GmbH, Jena, Germany).
[0053] 2.3.2 Molecular biological identification
[0054] Genomic DNA was extracted from the screened biocontrol strains using the Ezup column-based fungal genomic DNA extraction kit from Shanghai Sangon Biotech. ITS universal primer pairs (ITS4 and ITS5), RPB2 primer pairs (fRPB2-5f and fRPB2-7cr), and TEF1 primer pairs (EF1-728F and TEF1LLErev), synthesized by Beijing Qingke Biotechnology (Chongqing) Co., Ltd., were used for amplification. Specific primer information is shown in Table 1.
[0055] Table 1 Primer Information
[0056]
[0057] Polymerase chain reaction (PCR) was performed in a 25 μL reaction volume containing 1 μL DNA template, 1 μL forward primer, 1 μL reverse primer, 12.5 μL 2× Tap Plus MaserMix, and 9.5 μL sterile water.
[0058] The PCR products were purified and sequenced by Beijing Qingke Biotechnology Co., Ltd. (Chongqing).
[0059] RPB2 and TEF1-α sequences of 32 other Trichoderma strains and two outgroups, Protocrea farinosa CBS121551 and P. pallida CBS 299.78, were downloaded from GenBank (see Table 2). Phylogenetic trees were constructed using the maximum likelihood (ML) method. ML analysis was performed on the IQ-TREE web server (http: / / iqtree.cibiv.univie.ac.at) with 1000 fast guide replicates under a partitioning model. The phylogenetic trees were visualized using FigTree v.1.4.0.
[0060] Table 2. Strains' genetic information
[0061]
[0062] Note: The presence of T and ET in the strain number indicates that this strain is a type strain.
[0063] 3. Efficacy determination of biocontrol bacteria against detached fruits of kiwifruit soft rot
[0064] Preparation of biocontrol bacterial spore suspension: The screened biocontrol strains were inoculated onto PDA medium plates and incubated at 25℃ for 5 days. Subsequently, the culture was washed with sterile water to prepare a suspension with a concentration of 1×10⁻⁶. 6 Prepare a spore suspension at spore / mL.
[0065] The "Guichang" kiwifruit variety was selected. Healthy fruits with uniform shape, size, and ripeness were chosen. A puncture wound was made at the equator of the fruit, and a solution of 10% concentration was applied. 6 50 μL of a spore suspension of biocontrol bacteria (spores / mL) was added to the wound and allowed to air dry for 1 hour. Fresh soft rot fungal cakes (3 mm in diameter) were taken using a punch and placed on the kiwifruit wound. A positive control was inoculated only with pathogenic fungal cakes, and a negative control was inoculated with sterile PDA fungal cakes (added with sterile water). The treated fruits were placed in a 25℃ incubator to observe disease development and measure lesion diameter. The inoculation was repeated three times. Seven days after inoculation, the size of leaf lesions was measured using calipers. The disease control effect on detached kiwifruit soft rot was calculated using the following formula: Disease control effect (%) = [(control group lesion diameter - treatment group lesion diameter) / control group lesion diameter] × 100%.
[0066] 4. Determination of the antibacterial effect of volatile products of biocontrol bacteria
[0067] The antibacterial activity of volatile metabolites of screened biocontrol strains against *Botryosphaeria dothidea* was evaluated using the plate-on-plate method. Biocontrol strain and pathogen mycelial discs (5 mm in diameter) were inoculated into the center of PDA plates. The pathogen-inoculated plate was then inverted onto the biocontrol-inoculated plate, and the gap between the two plates was sealed. The control group was inoculated only with the pathogen and cultured in the same manner. All treatments were incubated at 25°C, with three biological replicates for each treatment. When the control pathogen colony had fully grown on the plate, the diameter of the pathogen colony in the treatment group was measured using the cross-cross method, and the inhibition rate was calculated as: inhibition rate = [(C-5) - (T-5) / (C-5)] × 100%. Here, C and T represent the diameters of the pathogens tested in the control and treatment groups, respectively.
[0068] 5. Broad-spectrum antibacterial activity of biocontrol bacteria
[0069] Eight different fungal pathogens (Fusarium solani, Fusarium graminearum, Fusarium asiaticum, Rhizoctonia solani, Sclerotium rolfsii, Colletotrichum musae, Diaporthephaseolorum, and Alternaria tenuissima) were selected, and the broad-spectrum inhibitory activity of the biocontrol strains was determined using the plate confrontation method. The inhibition rate was calculated according to "3. Method for determining the control efficacy of biocontrol bacteria against detached fruits of kiwifruit soft rot".
[0070] All eight fungal pathogens mentioned above were preserved by the Department of Plant Pathology, Guizhou University, Guiyang City, Guizhou Province.
[0071] 6. Results Analysis
[0072] 6.1 Isolation and Screening of Biocontrol Strains for Kiwifruit Soft Rot Disease
[0073] The inhibitory effect of Trichoderma strains on *Botryosphaeria dothidea* was determined using the plate confrontation method, with *Botryosphaeria dothidea* as the indicator fungus. Results are shown below. Figure 1 According to Table 3, a Trichoderma strain with good inhibitory effect on B. dothidea was finally screened and named GUDJ16, with an inhibition rate of 77.15%.
[0074] Table 3. Antimicrobial effects of biocontrol strains on Botryosphaeria dothidea pathogen.
[0075] deal with Colony size (mm) Inhibition rate CK <![CDATA[58.46±0.29 b ]]> - deal with <![CDATA[13.36±0.95 a ]]> 77.15±1.62
[0076] Note: Different letters in the table indicate significant differences (P < 0.05), as in the table below.
[0077] 6.2 Morphological identification
[0078] The strain GUDJ16, which showed good antibacterial effect against Botryosphaeria dothidea and was previously isolated and screened, was morphologically identified.
[0079] The strains were able to fully colonize plates on PDA, SNA, and CMA media after 5 days, and significant differences in colony morphology were found on different media.
[0080] On PDA medium, the colonies appear slightly cottony with dense hyphae. Green conidia form thick, dense concentric rings. The conidia are more concentrated and darker in color near the mycelial cake. The colonies produce no pigment and have a faint coconut aroma. On SNA medium, the colonies appear willow-like with sparse aerial hyphae, and conidia form thin, scattered concentric rings. The conidia are yellowish-green, turning green near the mycelial cake. On CMA medium, the aerial hyphae are abundant, and the conidia are thick and dense, covering the entire plate.
[0081] The strains produced conidiophores and conidia after 7 days of culture on PDA medium at 28°C. The conidiophores had a distinct main axis with paired or unilateral lateral branches arranged relatively closely. The flask-shaped phialides were long-necked, mostly whorled in groups of 2-3, occasionally solitary, and measured 5.09-11.10 μm × 2.3-4.31 μm. The conidia were unicellular, spherical, measuring 3.1-3.9 μm × 3.15-3.78 μm, and appeared pale yellowish-green under a microscope; no chlamydospores were present.
[0082] 6.3 Multigene phylogenetic analysis
[0083] Multilocus phylogenetic analysis was performed on the sequences of 35 strains, including those screened in this invention. The analysis used tandem sequences of the rpb2 and tef1 genes, with *Protocrea farinosa* CBS 121551 and *P. pallida* CBS 299.78 as outgroups. In the maximum likelihood (ML) phylogenetic tree, all strains formed monophyletic groups with high statistical support values, and species GUDJ16 formed a separate branch. Comparison revealed significant differences in colony morphology compared to *T. simile*, the absence of chlamydospores in GUDJ16, significant differences in colony morphology compared to *T. guizhouense*, and larger molecular spores in GUDJ16. Based on morphological and molecular biological identification, it was thus identified as a new species of *Trichoderma*—*Trichoderma rhododendron*.
[0084] Trichoderma rhododendron GUDJ16 was deposited at the China Center for Type Culture Collection (CCTCC) on September 11, 2025, at Wuhan University, Wuhan, China, with accession number CCTCC NO:M 20252004.
[0085] 6.4 Control effect of Trichoderma rhododendron GUDJ16 on detached fruits of kiwifruit soft rot
[0086] See results Figure 4 See Table 4. Seven days after treatment, no disease symptoms were observed in the fruits of the negative control group, while disease appeared in the fruits of both the positive control group and the treated group inoculated with the pathogen. The average diameter of lesions in the positive control group was 43.49 mm, while the average diameter in the treated group was 16.85 mm, showing a significant difference. The *Trichoderma rhombifolia* GUDJ16 screened in this invention showed a control efficacy of 61.26% against kiwifruit soft rot. Therefore, *Trichoderma rhombifolia* GUDJ16 obtained in this invention has excellent control efficacy against kiwifruit soft rot.
[0087] Table 4. Control effects of different treatment groups on detached kiwifruit soft rot.
[0088] deal with Lesion diameter (mm) Relative efficacy (%) negative control group - - Positive control group <![CDATA[43.49±1.77 b ]]> - GUDJ16 <![CDATA[16.85±0.09 a ]]> 61.26±0.21
[0089] 6.5 Antibacterial effect of volatile substances from Trichoderma rhododendron GUDJ16 on B. dothidea
[0090] The results showed that ( Figure 5 (As shown in Table 5), the volatile substances of Trichoderma rhododendron GUDJ16 have a significant inhibitory effect on the hyphal diameter of B. dothidea and also inhibit the sporulation of B. dothidea on PDA plates.
[0091] Table 5. Inhibition rate of volatile substances from Trichoderma rhododendron GUDJ16 on hyphal diameter of B. dothidea
[0092] Colony diameter (mm) Antibacterial rate (%) CK <![CDATA[83.65±0.35 b ]]> - GUDJ16 <![CDATA[52.75±0.41 a ]]> 39.23±0.52
[0093] 6.6 Broad-spectrum antibacterial activity of Trichoderma rhododendron GUDJ16
[0094] See results Figure 6 Through experiments on the inhibition of mycelial growth of eight pathogenic fungi, the results showed that Trichoderma rhododendron GUDJ16 had an inhibition rate of over 53% against all pathogens. Among them, the inhibition rate against Fusarium solani, Fusarium graminearum and Rhizoctonia solani was less than 60%, while the inhibition rate against the other five pathogens was over 65%.
[0095] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
Claims
1. A type of Trichoderma rhododendron (GUDJ16), characterized in that, The aforementioned Trichoderma rhodopsin GUDJ16 was deposited at the China Center for Type Culture Collection (CCTCC) on September 11, 2025, at Wuhan University, Wuhan, China, with accession number CCTCC NO:M 20252004.
2. A spore suspension, characterized in that, The spore suspension contains spores of Trichoderma rhododendron GUDJ16 as described in claim 1.
3. A method for preparing the spore suspension according to claim 2, characterized in that, The process includes the step of inoculating the *Trichoderma rhododendron* GUDJ16 into a culture medium and culturing it for 5 days, followed by eluting the culture to obtain the spore suspension.
4. The use of the Trichoderma rhododendron GUDJ16 as described in claim 1 or the spore suspension as described in claim 2 in the preparation of microbial inoculants.
5. A microbial inoculant, characterized in that, The microbial agent includes Trichoderma rhododendron GUDJ16 as described in claim 1 or the spore suspension as described in claim 2.
6. The application of Trichoderma rhododendron GUDJ16 as described in claim 1, the spore suspension as described in claim 2, or the microbial agent as described in claim 5 in the control of plant pathogens.
7. The application according to claim 6, characterized in that, The plant pathogens are selected from one or more of the following: Staphylococcus aureus, Fusarium solani, Fusarium graminearum, Fusarium asiaticum, Rhizoctonia solani, Sclerotium sclerotiorum, Banana anthracnose fungus, Bean intercropping scabies, and Alternaria spp.
8. The use of Trichoderma rhododendron GUDJ16 as described in claim 1, the spore suspension as described in claim 2, or the microbial agent as described in claim 5 in the preparation of products for controlling plant pathogens.
9. A product for preventing and controlling plant pathogens, characterized in that, The product includes Trichoderma rhododendron GUDJ16 as described in claim 1, the spore suspension as described in claim 2, or the microbial agent as described in claim 5; The plant pathogens are selected from one or more of the following: Staphylococcus aureus, Fusarium solani, Fusarium graminearum, Fusarium asiaticum, Rhizoctonia solani, Sclerotium sclerotiorum, Banana anthracnose fungus, Bean intercropping scabies, and Alternaria spp.
10. A method for controlling plant pathogens, characterized in that, The method includes the step of applying the Trichoderma rhododendron GUDJ16 of claim 1, the spore suspension of claim 2, or the microbial agent of claim 5 to the plant. The plant pathogens are selected from one or more of the following: Staphylococcus aureus, Fusarium solani, Fusarium graminearum, Fusarium asiaticum, Rhizoctonia solani, Sclerotium sclerotiorum, Banana anthracnose fungus, Bean intercropping scabies, and Alternaria spp.