Abstract:Objective Porcine epidemic diarrhea, caused by porcine epidemic diarrhea virus (PEDV), is a highly contagious viral disease and results in high mortality of pigs and huge lost of pig industry. The CRISPR/Cas13b system can mediate a highly efficient cleavage or editing to target RNA, thereby offering a novel strategy for interfering the infection of RNA viruses. We here tried to use the CRISPR/Cas13b system to cleave the PEDV RNA genome, in order to explore a novel strategy to inhibit PEDV infection.Method We designed four CRISPR RNA (crRNA) sequences which respectively recognize four regions in the PEDV genome. The CRISPR/Cas13b targeting vectors were constructed and transfected into Vero cells. The transfected cells were infected by PEDV, and then we analyzed the viral load of PEDV in cultured cells.Result The CRISPR/Cas13b system significantly inhibited PEDV propagation in Vero cells. In the viral immunofluorescence assay, the transfected cells with targeting vectors U6-crRNA3 and U6-crRNA4 had obviously fewer fluorophores compared with normal cells. The quantitative PCR results showed that CRISPR/Cas13b decreased PEDV load in cultured cells by above 50%.Conclusion The constructed CRISPR/Cas13b system can effectively interfere the propagation of PEDV. This study provides an alternative approach for effective RNA virus prevention and control, and creation of disease-resistant pig models.

Abstract:Objective To study the effect of pagP gene deletion on outer membrane characteristics of avian pathogenic Escherichia coli(APEC).Method The minimal inhibitory concentration (MIC) experiment was used to explore the effect of pagP deletion on the permeability of outer membrane of strain. Autoaggregation experiment, outer membrane hydrophobicity test, and analysis of biofilm-forming conditions of strain were performed to understand the influence of pagP deletion on biofilm-forming ability. The biofilm morphology was observed by scanning electron microscopy.Result The MIC of strain decreased after the deletion of pagP gene, the outer membrane permeability of strain increased, and the autoaggregation ability of strain increased significantly (P < 0.01). The MICs of erythromycin and ampicillin were 7 and 20 μg/mL respectively. The autoaggregation ability of strain was 87.89%. The deletion of pagP gene had no significant effect on the hydrophobicity of outer membrane of strain, and the hydrophobicity was only 5.337%. The amount of biofilm increased with the increase of time when the strain was cultured in LB medium. The strain with pagP deletion had higher biofilm-forming ability than wild strain.Conclusion The deletion of pagP gene can change the characteristics of outer membrane of APEC and enhance the biofilm-forming ability.

Abstract:Objective To investigate antimicrobial resistance and resistance genes of Escherichia coli from pet animals in Guangzhou.Method From July 2016 to July 2017, 319 samples were collected from cats and dogs at four animal hospitals in Guangzhou, including 127 samples from healthy animals and 192 samples from diseased animals. E. coli strains were isolated using the selective media, and were identified by MALDI-TOF mass spectrometry. The susceptibilities of E. coli isolates to 11 antimicrobial agents were determined by the agar dilution method. The presence of resistance genes was determined by PCR and sequencing.Result A total of 203 E. coli strains were isolated from 319 samples, including 109 E. coli isolates from diseased animals and 94 E. coli isolates from healthy animals. Among the 203 strains, 179 strains were resistant to at least one antimicrobial agent. The isolates showed the highest resistance rate against ampicillin (76.85%). The resistance rates against cefotaxime, tetracycline, doxycycline and sulfamethoxazole-trimethoprim were above 50%. The isolates were the most susceptible to amikacin with the resistance rate of only 10.84%. The isolates from diseased animals showed higher resistance rates against all antimicrobial agents compared with those from healthy animals. The resistance rates against all antimicrobial agents except amikacin, florfenicol and fosfomycin were significantly higher in the isolates from diseased animals compared with healthy animals (P < 0.01). The detection results of resistance genes showed that floR had the highest detection rate (34.97%), the detection rates of bla_CTX-M-9G, bla_CTX-M-1G, fosA3, rmtB and bla_CMY-2 genes were 22.66%, 20.19%, 17.73%,10.34% and 1.48% respectively. The genes bla_CTX-M-2G and bla_CTX-M-25G were not detected.Conclusion The antimicrobial resistance of E. coli isolates from pet animals in Guangzhou has become a serious problem, and some isolates carry several important resistance genes. Antimicrobial resistance in pet animals requires strict monitoring.

Abstract:Objective To perform etiological investigation of canine distemper virus (CDV) infection in dogs from Guangzhou and Dongguan, moniter the genetic evolution of canine CDV, and provide a data basis for the prevention and control of canine distemper (CD).Method Seventeen CDV positive samples were identified from dogs with CD symptoms. The haemagglutinin (H) genes of these wild strains were obtained by RT-PCR, and the H gene sequences were compared and analyzed through bioinformatics approach.Result The similarities of nucleotide and amino acid sequences of H genes from 17 CDV strains were 97.4% to 100.0% and 97.5% to 100.0%, respectively. Compared with vaccine strains such as Onderstepoort, Lederle and Convac, the similarities of nucleotide and amino acid sequences of H genes from these CDV strains were 90.3% to 91.5% and 89.4% to 90.8%, respectively. Phylogenetic tree analysis showed that 17 CDV wild strains belonged to Asia I subtype and were distant from the vaccine strains. The identified wild strains had evolved to containing nine potential N-glycosylation sites.Conclusion Asia I CDV is still an epidemic subtype in this region and this subtype is stable, but it has formed a certain evolutionary distance and a large number of mutations compared with vaccine strains. Therefore, it is of great significance to continue to monitor the evolution of CDV in dog populations.

Abstract:Objective To explore a new method for isolating proteins released by spermatozoa of pig epididymis, and determine the functional characteristics and signaling pathways of released proteins.Method The unique co-culture system of Transwell chamber was used to separate proteins secreted by sperms through 0.4 μm polycarbonate membrane. The isotopically labeled relative and absolute quantitation (iTRAQ) technique was used to analyze the released proteins and identify the differential proteins.The most important biochemical metabolic pathway and signal transduction pathway of differential proteins were confirmed by GO database describing their functions and KEGG database analyzing their pathways. Meanwhile, IPR database was used to conduct non-redundancy analysis for the domains of differential proteins.Result A total of 542 proteins were identified in the upper and lower rooms of Transwell chamber, of which 464 proteins were significantly up-regulated and 78 proteins were significantly down-regulated. The differential proteins were mainly involved in biological processes such as transmembrane transport, ion transport and ATP synthesis. The main involved signaling pathways of differential proteins were aldosterone-mediated sodium salt uptake, vesicle transport, and EGFR tyrosine kinase inhibition. Sulfur-oxidized protein, galactose, glycoside hydrolase and other major domains that constituted differential proteins provided structural bases for protein functions.Conclusion The method of separating proteins using Transwell chamber is effective. iTRAQ technique successfully identifies the differential proteins in proteins released by spermatozoa in epididymal head. The difference analysis and function annotation lay the foundation for exploring life activities and functional roles that differential proteins involved.

Abstract:Objective To explore the effects of rice-amaranth intercropping on rice yield, rhizosphere soil available nutrient and As accumulation in various parts of rice.Method Through field plot experiment, four treatments including rice monocropping (control), rice-white amaranth intercropping, rice-grain amaranth intercropping and rice-red amaranth intercropping were setted to study the changes of rice yield and As accumulation, rhizosphere soil available nutrient and organic matter contents.Result As contents of shoot, root, husk and brown rice during harvest time in four treatments all behaved as rice-white amaranth intercropping > rice-grain amaranth intercropping > rice-red amaranth intercropping > rice monocropping. Compared with the control, rice-white amaranth intercropping significantly increased As contents of shoot, root, husk and brown rice by 88.50%, 65.38%, 68.25%, 45.45% respectively. As extract amount from soil in rice-white amaranth intercropping treatment reached 5.44 mg·m^-2, which was 1.67 times significantly higher than control. The biomass per rice plant in rice-white amaranth intercropping treatment was significantly higher than control, rice-red amaranth intercropping and rice-grain amaranth intercropping, and 63.79% higher than control. Compared with control, intercropping treatments significantly reduced rice yield per unit area. Rice-white amaranth intercropping had the highest yield per unit area, which was 59.15% of control. The available nutrient and organic matter contents in rice rhizosphere soil increased in different degrees compared with monoculture.Conclusion Rice-amaranth intercropping reduces rice yield per unit area, and significantly increases As contents in shoot, root, husk and brown rice, and available nutrient and organic matter contents in rhizosphere soil. Rice-white amaranth intercropping is the optimal scheme, as it extract the most As in As contaminated soil, and the grown amaranth meets national safety standards.

Abstract:Objective To explore the differences in rice yield compositions and material productions among different cultivation modes in soda saline-alkali rice area of the western Songnen Plain, and increase local rice yield.Method Taking ‘Jijing 88’ as the test material, four cultivation modes were setted, including no fertilization (basic), local farmer cultivation (control), high yield and high efficiency cultivation and super high yield cultivation. The yield formations and material production characteristics of rice under four modes were compared and analyzed.Result High yield and high efficiency cultivation and super high yield cultivation significantly increased dry matter accumulations from jointing stage to maturity stage, with the average yields in two years of 10.34 and 12.15 t·hm^-2 respectively. Compared with the control, the yields increased by 8.44% and 27.45% respectively, the effective panicles increased by 3.03% and 34.10% respectively, and the numbers of spikelets per panicle increased by 4.06% and 9.37% respectively.Conclusion The integrated and optimized cultivation techniques promote material production and transportation, optimize panicle structure, increase the numbers of effective panicle and spikelets per panicle, and greatly increase rice yield in soda saline-alkaline rice area of the western Songnen Plain.

Abstract:Objective To explore the drought resistances of 10 fine sugarcane lines and provide references for breeding, popularization and application of drought resistant cultivars.Method Changes of sugarcane root vitality, leaf proline and soluble protein and soluble sugar contents, leaf superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) activities at early elongation stage were measured under artificial drought stress and normal water supply treatments. Principal component analysis and cluster analysis were used to comprehensively evaluate the drought resistances of sugarcane lines at early elongation stage.Result Under drought stress, root activities of tested sugarcane lines decreased, while the contents of leaf soluble protein, soluble sugar, proline and malondialdehyde increased, as well as the activities of leaf SOD, POD and CAT. Cluster analysis divided 11 sugarcane lines including the control cultivar ROC22 into three categories, including strong drought resistance, moderate drought resistance and weak drought resistance. New sugarcane lines with strong drought resistance were A6-13115 and A3-1320, with moderate drought resistance were A13-1396 and A6-13122, with weak drought resistance were A6-13111, A11-1390, A1-1305, A7-13120, A7-13104 and A4-1316.Conclusion Using principal component analysis and cluster analysis, the comprehensive evaluation of multiple indicators, and can improve the reliability and accuracy of sugarcane drought resistance evaluation. New sugarcane lines A6-13115 and A3-1320 have strong drought resistance.

Abstract:Objective To study the characteristics of nitrogen(N), phosphorus(P) and potassium (K) nutrient release and soil N, P, K nutrient balance under straw mulching pattern during potato planting process.Method We studied the decomposition rate and nutrient release status of mulching straw using pot experiment.Result Straw carbon (C) and P contents changed slightly after mulching for 82 days, while K content decreased by 93.96%, and N content increased by 49.22%. The mulching straw provided a large amount of nutrients (especially K) with 100 kg mulching straw contributing 0.42 kg N, 0.14 kg P (P₂O₅), and 1.70 kg K (K₂O) to soil used for potato growth. Straw decomposition rate was 70.87%. The nutrient accumulative release rates were performed as: K (98.22%) > C (71.89%) > P (68.75%) > N (56.85%). Straw mulching and K fertilization largely affected soil K balance. The K deficient amount was 163.80 kg/hm² without straw mulching and K fertilizer application. K deficient amount was effectively reduced by straw mulching and K fertilization, and even K had surplus with combined application of straw and K fertilizer.Conclusion The straw mulching pattern can reduce K fertilizer application amount and production cost, and relieve the dilemma of K fertilizer resource deficiency in China.

Abstract:Objective To reduce veterinary antibiotic residues in domestic sewage and livestock breeding wastewater, and screen aquatic plants which can better remove veterinary antibiotics in water for plant restoration and artificial wetland.Method Four commonly used aquatic plants (Pennisetum hydridum, Cyperus alternifolius, Canna indica and Pontderia cordata) and three kinds of commonly used veterinary antibiotics (amoxicillin, florfenicol and doxycycline hydrochloride) were selected to construct the hydroponic experiment system for evaluating the tolerance and removal efficiency of antibiotics in water.Result Four different aquatic plant species had a certain efficiency on removing the selected veterinary antibiotics after 14 days of hydroponic culture. Three kinds of antibiotics had a positive effect on increasing plant height and chlorophyll content at low and medium concentrations of 0-100 mg/L, and inhibited plant growth and decreased chlorophyll content at high concentration above 100 mg/L. Besides, there was significant difference between the treatment groups and the control group. The highest removal efficiency was observed for each plant at 14 days after 100 mg/L antibiotics stress treatment. P. hydridum, C. indica and C. indica were the best selected plants to remove amoxicillin, florfenicol, and doxycycline hydrochloride respectively, and the removal rates were 55.0%, 56.2% and 48.3% respectively.Conclusion The efficiency of four kinds of plants to remove veterinary antibiotics in water was in order of C. plantsiana > P. hydridum > P. cordata=C. alternifolius.

Abstract:Objective To study the number and distribution characteristics of 45S and 5S rDNA sequences in the genome of 20 species of Cucurbitaceae plants，and provide a basis for studying the karyotype, genetic breeding and evolutionary classification of Cucurbitaceae plants.Method Using improved fluorescence in situ hybridization (FISH) method, the 45S and 5S rDNA probe sequences were modified by fluorescence at the 5' ends, and the physical locations of 45S and 5S rDNA in the metaphase chromosomes of 20 species of Cucurbitaceae plants were determined. The chromosomes were observed under the Nikon 80i fluorescence microscope, and images were collected by cold CCD and then were analyzed.Result Numbers, locations and characteristics of 45S rDNA and 5S rDNA fluorescence sites on the metaphase chromosomes of 12 plants including Gymnopetalum chinense, Herpetospermum pedunculosum, Lagenaria siceraria, Momordica cochinchinensis, Momordica dioica, Cucurbita pepo, Trichosanthes anguina, Trichosanthes dunniana, Trichosanthes ovigera, Zehneria maysorensis, Coccinia grandis and Sechium edulewere were defined. We detected 3, 7, 2, 4, 2, 5, 3, 3, 5, 1, 2 and 2 pairs of 45S rDNA from these 12 plants respectively, and detected 2, 1, 1, 1, 1, 2, 1, 1, 1, 1, 1 and 1 pairs of 5S rDNA respectively. The 45S rDNA and 5S rDNA of 20 plants were found to be located on short arms of chromosomes, terminal ends of short arms or centromeres.Conclusion FISH is an effective tool for building fine karyotypes of Cucurbitaceae plants. It can help to analyze the random body, differentiate chromosome and identify homologous chromosomes, and therefore provides strong evidence for karyotype analysis.

Abstract:Objective To investigate the effects of litchi polyphenols on growth and development of Peronophythora litchii.Method Polyphenols were extracted from litchi pericarp by solvent extraction. The effects of polyphenols on colony morphology, biomass, germination mode of sporangium, zoospore germination and oospore number of P. litchii were analyzed by phytopathological research methods.Result With the treatment of litchi polyphenols at 150 to 300 μg·mL^-1, the hyphal colonies of P. litchii appeared dense, hyphal dry weight and sporangia number were significantly reduced, and the sporangia germinated to release zoospores. With the treatment of litchi polyphenols at 100 to 300 μg·mL^-1, zoospores germination was significantly increased while sporangia germination was significantly suppressed. Oospore was hardly observed in PDA control without litchi polyphenols. When litchi polyphenols in the media reached 100 μg·mL^-1, the produce of oospores was 10.8 mm^-2.Conclusion Litchi polyphenols involve in the regulation of the growth and development of P. litchii, can suppress the growth of P. litchii and sporangia germination, indicating their potential for developing new safe biogenic fungicides.

Abstract:Objective Damages in subtropical regions of China caused by forest pests have been intensified due to environmental changes such as global warming. It is of great importance to study the relationship between leaf physiological traits and pest infestation grade.Method The relationships of main physiological traits, including non-structural substance contents and enzyme activities, and pest infestation grade on healthy leaves of Machilus pauhoi and Phoebe bournei artificial mixed forest were studied by typical plot survey.Result Pest damage caused significant increase in the relative water contents of healthy leaves of affected individuals, while did not have significant effect on leaf dry matter content. The soluble sugar contents of healthy leaves of M. pauhoi and P. bournei were the lowest in trees with moderate pest damage. The soluble protein content decreased in M. pauhoi healthy leaves with the increase of pest infestation grade. The soluble protein content of healthy leaves of P. bournei was the highest with slight pest damage. Total amino acid contents of healthy leaves of P. bournei and M. pauhoi were the highest with moderate pest damage and slight pest damage respectively. The ratios of soluble protein conents to soluble sugar contents reached the maximum in trees with moderate pest damage for both species, but the lowest values appeared in different pest levels for two species. The ratio of soluble protein content to soluble sugar content in leaves of P. bournei was significantly different between slight pest and moderate pest infestation grade. The peroxidase (POD) activities of healthy leaves of two species generally decreased first and then increased with the increase of pest infestation grade. The superoxide dismutase (SOD) activities were the highest under moderate pest infestation grade and the lowest under severe pest infestation grade, yet the differences among different pest infestation grade were not significant.Conclusion Two tree species have different response in non-structural substance contents to the same infestation grade of pest, while they have the same response in enzyme activities to the same infestation grade of pest. The damaged leaves and healthy leaves of two species have mutual compensation and self-regulation, and both species have self-healing ability and defense mechanism. The research results can provide a theoretical basis for further exploring the response mechanism of plant leaves to pests under future environmental changes and carrying out research on pest control techniques for artificial forest.

Abstract:Objective To explore the carbon storage of Sonneratia apetala in Guangdong Province, provide basic data for carbon sequestration investigation and monitoring based on mangrove biomass in Guangdong Province, and provide experiences and methods for monitoring mangrove carbon sequestration in China.Method S. apetala vegetation and 0−100 cm soil were taken as research objects to build biomass model that could be universally used in Guangdong Province, and compare carbon storage of vegetation and soil in ten regions.Result The biomass model of S. apetala was W=0.033(D²H)^1.002, and the determination coefficient was 0.952. The fitting effect of the model was good. Total area of S. apetala forest in Guangdong Province was 1 724.12 hm², total carbon storage was 536 801.09 t, vegetation carbon density was 50.81 t·hm^-2, soil carbon density was 260.54 t·hm^-2 and total carbon density was 311.35 t·hm^-2. Vegetation carbon density was 16.32% of total carbon density, and soil carbon density was 83.68% of total carbon density. Total carbon reserves of S. apetala forest in ten regions were performed in the order of Shenzhen 2 790.65 t < Chaozhou 3 088.34 t < Huizhou 10 479.30 t < Jiangmen 13 800.58 t < Maoming 17 116.43 t < Zhanjiang 55 610.15 t < Zhongshan 58 562.90 t < Shantou 66 498.62 t < Guangzhou 134 938.18 t < Zhuhai 173 915.93 t.Conclusion Carbon storage of S. apetala in Guangdong Province is mainly concentrated in soil layer. Soil carbon storage and vegetation carbon storage of S. apetala are obviously different in different regions.

Abstract:Objective To explore the responses of soil available nutrients to different tending measures and seasonal changes of the nutrient contents, select the tending measures that could effectively improve soil nutrient status of woodland, and provide references for tending system establishment of mixed plantation of Dalbergia odorifera and Santalum album.Method The surface soil of the mixed plantation of D. odorifera and S. album was studied. The experiment setted four treatments, including control (CK), weeding, fertilization, weeding+fertilization. Each treatment was repeated four times. Changes of soil available nutrient contents and pH values were measured quarterly.Result Weeding and fertilization did not significantly change soil pH, but enhanced acid-base buffering performance of soil in some extent, and slowed the seasonal fluctuation of soil pH value. The soil available nutrient contents were significantly affected by season and tending measure changes. The contents of ammonium nitrogen and nitrate nitrogen in four treatments peaked in autumn and winter. Compared to CK, the annual ammonium nitrogen contents of soil in weeding, fertilization and weeding+fertilization treatments increased by 16.87%, 8.17% and 20.25% respectively. Soil nitrate nitrogen contents of weeding and weeding+fertilization treatments increased by 12.69% and 21.45% respectively compared with CK, while the content of fertilization treatment decreased by 1.81%. Soil available phosphorus contents of fertilization and weeding+fertilization treatments increased by 19.58% and 50.63% respectively, while the content of weeding treatment decreased by 2.53%. Soil available potassium contents of weeding, fertilization and weeding+fertilization treatments increased by 117.18%, 57.91% and 142.54% respectively.Conclusion The soil available nutrients have different responses to tending measures. Weeding and fertilization increases the content of one or more available nutrients in soil, while weeding+fertilization promotes overall increases of soil available nutrient contents under the forest. The weeding+fertilization treatment is an ideal tending measure for D. odorifera and S. album mixed plantation, and it is expected to promote the tree growth well.

Abstract:Objective To explore the effects of different fertilization ratios on compost humus composition.Method Pholiota nameko residue was selected as the basic material. Different proportions of pig manure were added to analyze humus structure, and screen the best ratio of P. nameko residue to pig manure while composting organic fertilizer.Result In the compost with different ratios of P. nameko residue to pig manure, the contents of water soluble organic matter and humic acid carbon all decreased firstly and then increased slowly as the composting process continued. The ratios of humic acid to fulvic acid (C_HA/ C_FA) all decreased significantly on the 15th day, and then increased. C_HA/C_FA in treatment Ⅰ, Ⅱ, Ⅲ, Ⅳ and Ⅴ(the mass ratios of P. nameko residue to pig manure were 0:10, 1:9, 3:7, 5:5 and 7:3) were significantly higher than the initial values, being 7.14, 3.23, 1.22, 4.38 and 2.52 times of the initial values respectively. C_HA/ C_FA in treatment Ⅳ was the best level. Meanwhile, the carbon content of humin in treatment Ⅳ was higher than those in other treatments.Conclusion In compost with different proportions of P. nameko residue and pig manure, the organic carbon contents are more beneficial, and the humus composition contents and soil fertility are improved, as compared with using P. nameko residue or pig manure alone. Under the appropriate proportions of P. nameko residue and pig manure, the polymerization and humification degrees of soil humus improve with the increase of C_HA/C_FA. Organic material can improve the humus content of each combination form and the structure of soil organic matter, and the effect is better than other treatments when the ratio of P. nameko residue to pig manure is 5:5.

Abstract:Objective To investigate interactions differences between L-tryptophan (L-Trp), D-tryptophan (D-Trp) enantiomers and polydopamine (PDA), and elucidate the chiral recognition mechanism in molecular imprinting.Method Computational molecular simulation was used for studing the molecularly imprinted pre-polymerization system with tryptophan as template and PDA as functional monomer. Firstly, conformational searching was used to obtain the potential stable structure of PDA tetramer. Then, molecular docking was employed to identify the interaction between Trp enantiomers and PDA. Finally, Quantum-chemical calculation was used to reveal the reason for the binding difference between the enantiomers and PDA from the perspective of electronic structure.Result Hydrogen bonding played a predominate role in the binding between Trp and PDA. The hydrogen bonding force between L-Trp and PDA was stronger than that between D-Trp and PDA. On the other hand, the frontier orbital Energy gap of L-Trp-PDA complex was bigger than that of D-Trp-PDA complex, and the former complex had tighter binding as well as stronger weak interaction.Conclusion Molecular simulation method is easy and rapid to operate, and it’s suitable for studing the chiral recognition mechanism in molecular imprinting.

Abstract:Objective To solve the insufficient identification of fine-grained tomato diseases in greenhouse.Method Taking tomato leaves with five early or late diseases as research objects, we proposed a new convolutional neural network model ARNet based on the combination of attention and residual thought. A multi-layered attention module was introduced to solve the problem of early disease location dispersion and the difficulty of feature extraction by extracting hierarchically disease classification information. In order to avoid the degradation of network training, we constructed a residual module to effectively integrate high- and low-order features. Meantime，we introduced the data expansion technology to prevent model over-fitting.Result Model training and testing results of early and late disease leaf datasets with 44 295 pictures showed that ARNet has better classification performance with an average recognition accuracy of 88.2%, which was significantly higher than those of other existing models. In addition, the identification accuracy of ARNet for early disease was significantly better than that for late disease, which verified the effectiveness of attention mechanism in extracting fine region features, and there was no excessive jitter during training process.Conclusion This model proposed in this paper has strong robustness and high stability, and can provide a reference for intelligent diagnosis of fine-grained tomato diseases in practical application.