Cucumber bacterial angular leaf spot, a global bacterial disease, has caused significant economic losses to the cucumber industry. Ventilation mode, temperature, humidity and other environmental factors in the greenhouse are important factors that affect the diffusion and transmission of pathogens. In this study, the impact of different ventilation methods in plastic sheds on the distribution and variation of temperature and humidity inside the greenhouse, as well as the diffusion of pathogens and disease transmission were evaluated.

Three ventilation methods (20.0 m×7.0 m×3.0 m) were set up in this experiment, including bottom and top ventilation, top ventilation, and bottom ventilation. After atomizing the bacterial suspension of Pal pathogens manually at the central position of the plastic shed for 0.5, 1.5, and 2.5 h, nutrient agar plates were used to collect Pal pathogens at distances of 0, 0.5, 1.0, 1.5, 2.0, 3.0, and 4.0 m from the center of the inoculum source. Based on the results of colony counts, a dynamic diagram of the deposition and diffusion of Pal pathogens was drawn to analyze the impact of ventilation modes on pathogen diffusion. An inoculation area for Pal pathogens was set up in the central location of the plastic shed. The pathogen sampler for agricultural cultivation space was used to collect Pal aerosols at distances of 0, 0.5, and 1.5 m from the inoculation area. Next, qPCR technology was adopted to detect the concentration of Pal pathogens, while the disease index of cucumbers was also investigated. Temperature and humidity recorders were used to monitor temperature and humidity data at different locations inside the cucumber greenhouse. Then, temperature and humidity distribution maps under different ventilation modes in the longitudinal and cross sections of the greenhouse were drawn and the impact of ventilation methods on the distribution and changes of temperature and humidity inside the greenhouse was analyzed.

The different ventilation methods in plastic sheds had a significant impact on the spread of Pal pathogens. With bottom and top ventilation, the concentration of Pal pathogens at different locations within the plastic shed was lowest, and the colony count was 3-469 CFU/m²; whereas with only bottom ventilation or top ventilation, there was local accumulation and more deposition of the pathogens, with the colony counts being 2-847 and 5-800 CFU/m², respectively. In field experiments on cucumber cultivation in plastic sheds, ventilation methods significantly affected the regulation of temperature and humidity inside the greenhouse as well as the transmission efficiency of cucumber bacterial angular leaf spot. Among them, the bottom and top ventilation treatment resulted in the best overall cooling and dehumidification effect inside the plastic sheds, with the lowest relative humidity at night. The humidity of the longitudinal and cross sections inside the greenhouse at 24: 00 were 70.6%RH-83.7%RH and 80.3%RH-93.0%RH, respectively, which were lower than those under the bottom ventilation treatment (80.2%RH-87.3%RH and 91.1%RH-96.1%RH) and top ventilation treatment (78.2%RH-92.2%RH and 91.9%RH-96.4%RH). Additionally, the concentration of Pal pathogens was the lowest at 8 128 CFU/m³, leading to the lightest disease incidence of cucumbers with a disease index of 23.33. The top ventilation treatment was the second best, with a concentration of Pal pathogens at 13 542 CFU/m³ and a cucumber disease index of 27.39. For the bottom ventilation treatment, the concentration of Pal pathogens was the highest, reaching 27 954 CFU/m³, resulting in the most severe disease incidence of cucumbers, with a disease index of 32.96.

The bottom and top ventilation strategy in plastic sheds has a good effect on cooling and dehumidifying the greenhouse. The spread of Pal pathogens is slow, resulting in mild disease incidence in cucumbers. The results of this study can provide a basis for guiding ventilation regulation in cucumber cultivation.

Long-term continuous cropping of tomato in Cangnan, Zhejiang Province leads to a series of soil continuous cropping obstacles, such as soil acidification, secondary salinization, soil compaction and serious soil-borne diseases. In this paper, the effects of tomato-rice rotation on soil physicochemical properties, enzyme activity, microbial biomass and microbial community structure under long-term continuous cropping were analyzed, aiming to provide a method to alleviate soil continuous cropping obstacles and improve soil environment.

Three treatments were set up and soil samples were collected. T1 was tomato continuous monoculture for 18 years, T2 was tomato continuous monoculture for 17 years followed by one year of tomato-rice rotation, and T3 was tomato continuous monoculture for 15 years followed by three years of tomato-rice rotation. Soil pH and EC values were measured by pH meter and conductivity meter. Soil physicochemical properties such as total carbon, total nitrogen and ammonium nitrogen were determined by high temperature combustion method, Kjeldahl nitrogen fixation method and potassium chloride solution leaching method. Soil enzyme activities were determined using enzyme activity kits. Total and harmful microorganism concentrations were detected using qPCR, and soil culturable microorganisms were counted using selective medium plate smear counting method. The MiSeq PE3000 high-throughput sequencing platform was used for sequencing, splicing and assembly, sequence comparison and functional annotation of the soil microgenome.

There were significant differences in soil physicochemical properties among different treatments. One and three years of tomato-rice rotation treatments increased soil pH from 5.20 to 6.04 and 6.73, and reduced EC from 558 μS·cm^-1 to 417 and 445 μS·cm^-1, and increased C∶N ratios from 9.16 to 10.45 and 10.74. Soil enzyme activities were increased in the rotations, with catalase, urease, polyphenol oxidase activities increased from 11.72 μmol·d^-1·g^-1, 10.76 μg·d^-1·g^-1, and 22.67 mg·d^-1·g^-1 to 58.58 μmol·d^-1·g^-1, 142.48 μg·d^-1·g^-1, and 37.10 mg·d^-1·g^-1. After crop rotation, the microbial population and community structure changed, harmful microorganisms decreased. The content of soil culturable actinomycetes increased, the fungi/bacteria ratio decreased. Chloroflexi, Acidobacteria, and Proteobacteria increased significantly, and Actinobacteria decreased significantly. The contents of Ralstonia solanacearum, Pectobacterium carotovorum subsp. carotovorum, P. c. subsp. brasiliensis and Fusarium sp. decreased to 1.76×10³, 7.28×10², 3.94×10³ and 3.07×10³ copies/g in the three years of crop rotation. The potential functions of soil microorganisms changed after crop rotation, with an increase in the abundance of genes related to carbohydrate metabolism, energy metabolism, and other related pathways. Specifically, genes associated with carbohydrate metabolism in metabolism were up-regulated.

Tomato-rice rotation can improve soil acidification, salinization and soil element imbalance caused by long-term monoculture, increase soil C∶N ratio and enzyme activity, reduce the occurrence of soil-borne diseases, transform the soil from fungal type to bacterial type, change the structure of soil microbial community, and promote soil microbial carbohydrate metabolism, which is important for improving soil continuous cropping obstacles.

Plasmodiophora brassicae is an obligate endoparasite that causes clubroot disease, which is the most devastating soil-borne disease in brassica crops. The propidium monoazide xx (PMAxx) could selectively bind to the chromosomal DNA of dead spores and therefore block DNA amplification by real-time fluorescent quantitative PCR (qPCR). In the present study, a strategy involving a PMAxx pre-treatment followed by the qPCR (PMAxx-qPCR) assay was developed for quantifying viable spores of P. brassicae, so as to provide a basis for early detection and prevention measurement of cloobroot disease.

PMA and PMAxx with concentrations of 0, 5, 10, 20, 40 and 60 µmol·L^-1 were prepared, respectively, and were used to pre-treat P. brassicae prior to DNA extraction, followed by qPCR. The inhibitory effects of PMA and PMAxx on DNA amplification of P. brassicae dead spores were compared, and the optimal nucleic acid dye and concentration to distinguish between live and dead spores were determined. The illumination time was set as 0, 2, 5, 10, 15 and 20 min, respectively, and the optimal exposure time was optimized to establish a PMAxx-qPCR assay for selectively detection of viable spores of P. brassicae. The mixed suspensions with different ratios of dead and viable spores (0, 0.01%, 0.1%, 1%, 10%, 25%, 50%, 75% and 100% viable spores) were prepared to determine the suitability of PMAxx-qPCR assay for distinguishing viable and dead spores. The assay was also applied to quantitative detection of viable spores of P. brassicae in 25 field soil samples.

PMAxx showed a better discrimination effect than PMA on the viable and dead spores of P. brassicae. When the concentration of P. brassicae was 1×10⁸ spores/mL, the optimal PMAxx concentration and light exposure time were 4 μmol·L^-1 and 10 min, respectively. The amplification of dead spores could be inhibited effectively, and only the DNA of living spores was targeted for selective amplification. For pre-defined ratio of viable spores, there was a good linear relationship between the lg of the P. brassicae DNA concentration assessed by PMAxx-qPCR and the theoretical viability (R²=0.992). For soil samples, viable P. brassicae was quantified in 11 of 25 samples, with infestation levels of approximately 32.35-6.97×10³ fg·g^-1.

The established method could quantitatively detect the viable spores of P. brassicae, with advantages of rapid, efficiency and sensitivity, which could be useful for avoiding the inability of qPCR method to distinguish between viable and nonviable spores. Application of the assay may potentially improve P. brassicae control and disease management.

Cucumber target leaf spot, caused by Corynespora cassiicola, has brought great economic losses to the cucumber industry. Sporulation and release of C. cassiicola spores play a significant role in the epidemiology of the disease. In this study, the regularity and the effect of humidity on sporulation and release of C. cassiicola, and the optimal application method and time for control of cucumber target leaf spot were evaluated.

The sporulation regularity of C. cassiicola was evaluated by quantifying the spore concentration on lesions of diseased cucumber leaves at 0: 00, 3: 00, 6: 00, 9: 00, 12: 00, 15: 00, 18: 00, and 21: 00, respectively. The release regularity of C. cassiicola was evaluated in different seasons of spring, summer, autumn and winter, air samples were collected from naturally infested cucumber greenhouse at 0: 00, 3: 00, 6: 00, 9: 00, 12: 00, 15: 00, 18: 00, and 21: 00, respectively, and the concentrations of C. cassiicola in the air were evaluated. The effect of relative humidity on sporulation and release of C. cassiicola was also assessed in artificial climate exposure chambers and plastic greenhouses at four different humidity conditions of continuous high humidity (RH>90%, 24 h), continuous low humidity (RH<60%, 24 h), high humidity for 12 h followed by low humidity for 12 h, and low humidity for 12 h followed by high humidity for 12 h. The control efficiency of 60% carbendazim·diethofencarb wettable powder (WP) and 500 million spores/g Pseudomonas fluorescens WP on cucumber target leaf spot disease was compared, by using powder spraying and water spraying at different application times in the field.

The study on the daily variation regularity of sporulation and release of C. cassiicola showed significant differences in the quantity of spores on diseased leaves and in the greenhouse air at different times of the day. There was a complementary relationship between the number of spores on diseased leaves and in the greenhouse air at the same time. After 18: 00, as the duration of high humidity (RH>90%) prolonged, the number of spores on diseased leaves increased, reaching a peak of 1 344 spores/cm² at 6: 00 the next day. Then, the humidity decreased gradually to RH<60% after opening the air vent of the greenhouse, and spores were released into the greenhouse space. At 12: 00, the spore concentrations in the greenhouse air reached a peak of 12 445-110 697 spores/m³. In different seasons of spring, summer, autumn, and winter, the daily variation regularity of sporulation and release is consistent, showing that C. cassiicola produced a large amount of spores under high humidity (RH>90%) at night, and released to greenhouses under low humidity (RH<60%) during the day. In artificial climate exposure chambers and plastic greenhouses, the highest quantity of C. cassiicola spores was detected under alternating wet and dry conditions, which was significantly higher than that under continuous high humidity or continuous low humidity condition. By powder spraying at 19: 00, 60% carbendazim·diethofencarb WP and 500 million spores/g P. fluorescens WP gave the best control efficiency of 80.60% and 75.08%, respectively, and the spore inhibition efficiency was higher than 84%.

Humidity is a key environmental factor affecting the spore reproduction and release of C. cassiicola. The alternating day-dry and night-wet environment in the greenhouse promotes the reproduction and diffusion of C. cassiicola, and accelerates the spread of cucumber target leaf spot. Powder spraying method is better than water spraying method for disease control, and the best application time is the evening before spore reproduction. The results of this study will contribute to the development of new strategies for the effective alleviation and control of cucumber target leaf spot.