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Root rot is a serious disease of garlic in the Shandong Province of China. Infected plants display symptoms such as dwarfed garlic bulbs, yellowing of leaves starting from the periole, discoloration of the basal stem, and rot. The aim of this study was the identification of the main pathogens involved in garlic root rot. Fifty garlic plants with root rot were collected from the five main production areas of garlic in the Shandong Province. Isolates was incubated using selective medium and identified using morphological method and the ribosomal DNA internal transcribed spacer (ITS) sequence alignment, followed by the confirmation of pathogenicity and the identification of biological characteristics of the putative pathogens. The isolation frequency of Pythium isolated from garlic root was 45%, resulting in a total of 12 isolates. According to morphological identification and ITS sequence alignment, the 12 isolates were further identified as Pythium sylvaticum Compbell & Hendrix, Pythium heterothallicum Compbell & Hendrix, Pythium paroecandrum Drechsler, and Pythium violae Chesters & Hickman. All 12 isolates were pathogenic when inoculated on the root collar and roots of garlic plants. The optimal temperature of Pythium growth was 20–25 °C and pH was 6–7. Pythium sylvaticum, Pythium heterothallicum, Pythium paroecandrum, Pythium violae, and Pythium sp. 845 us-2 were identified as causal agents of garlic root rot.

Head lettuce (Lactuca sativa L.) is an important crop for fresh consumption in China. In Shandong Province, head lettuce is planted in spring and in autumn each year. Because of the on-and-off rain for three weeks, head lettuce plants planted directly into the field in Jiyang City, in July 2017, 20% of the plants rapidly showed symptoms of rotting, water-soaked lesions on roots and stem bases, and then death. The diseased plants first appeared in low-lying areas prone to water accumulation. One-millimeter pieces were excised from water-soaked roots and stem bases, dipped in a 0.2% calcium hypochlorite solution for 10 min, then placed on V8 medium, and incubated in the dark at 28°C for 5 d. Two Pythium-like strains were isolated from the roots and stems. The isolates transferred to CMA and grown for 7 d, and the morphological characteristics of the two isolates on corn meal agar (CMA) were white with dense, cottony, aerial and well-branched mycelia. The two isolates produced sporangia, oogonia, antheridia and oospores. Most of the sporangia were lobate. The oogonia were smooth, nearly globose and terminal. Oospores were globose, smooth and aplerotic. The average dimensions of 50 oogonia and oospores respectively ranged from 19.5 to 25.2 (av. 23.1) µm and 17.8 to 22.3 (av. 19.9) µm. The antheridia were broadly sac-shaped. The isolates morphological characteristics were consistent with P. aphanidermatum (van der Plaats-Niterink, 1981). The COI gene and ITS region of the rDNA were amplified and sequenced using primers FM55/FM52R (Long et al. 2012) and ITS1/ITS4 (White et al. 1990), respectively. The two aligned COI sequences were identical for both isolates, as were the two ITS sequences. BLASTn analysis of the 1,133-bp COI sequence (accession no. MT952703) resulted in a 100% identity with accession number AY129164 from Lactuca sativa, which belongs to P. aphanidermatum, and the 808-bp ITS sequence (accession no. MT921597) showed a 99% identity with Genbank accession number HQ643442 belonging to P. aphanidermatum. Koch's postulates were conducted by first soaking corn kernels for 24 h in water, and then autoclaving for 2 h at 121˚C. Isolate SDHL-1 was grown on CMA for 10 days, after which agar plugs were transferred to the sterilized corn kernels and incubated at 28℃ for approximately 15 d, until the corn kernels were covered in white hyphae. Ten healthy head lettuce plants were transplanted into a sterilized loam potting soil artificially infested with the corn inoculum (3 g inoculum per 100 g loam mixture). Inoculated plants and noninoculated controls were maintained in a greenhouse at 28°C and 100% relative humidity with a 12-h photoperiod; the experiment was repeated once. All twenty inoculated plants exhibited symptoms within one week similar to those observed. Pythium aphanidermatum was recovered only from the water-soaked roots and stem bases of inoculated plants and the re-isolated cultures again identified based on morphological characteristics and sequencing of the ITS and COI genes. No symptoms were observed on the control plants. Sclerotinia sclerotiorum is reported to cause stem base rot of L. sativa in China (Zhou et al. 2011). To our knowledge, however, this is the first report of root rot of head lettuce caused by Pythium aphanidermatum. Identification of the pathogen will assist in devising strategies to reduce yield loss.

A new species, Dendrographium multiseptatum, is described and illustrated from specimens collected on rotten twigs in China. This fungus is characterized by synnematous conidiophores with polytretic conidiogenous cells producing obclavate, 6–9-distoseptate, pale brown conidia.

American ginseng (Panax quinquefolius) is a perennial herb whose dried roots are used for health care products, medicine, and food in China (Yuan et al. 2010). Shandong Province is the main area growing American ginseng and contributes more than 50% of the production in China. Wendeng city, located in the east of Shandong Peninsula, is the primary production area of American ginseng in Shandong Province since it has four distinct seasons, sufficient light, loose soil (pH 5.5～7.0), and with thus a similar geographical environment and climate conditions to the American ginseng production area of the United States and Canada. In March 2016, 2-year old American ginseng plants that were planted directly into the ground in the greenhouses in Wendeng city, contained up to 6-10% stunted plants. Water-soaked lesions were observed on the crowns and the tips of fine roots. The leaves of the infected plants became scalded, dark green starting at the top of the plants and gradually move downward. Moreover, the leaves and petioles gradually curled withered and drooped, and the whole plant collapsed. Tissue samples, 10 mm in size, were excised from the water-soaked roots and crowns of diseased plants, rinsed under running water for 24 hours, dipped in a 0.2% calcium hypochlorite solution for 10 minutes, placed on sterile filter paper to dry and then placed on V8 medium (200 mL V8 Campbell Soup, 15 g agar, 0.2 g CaCO3, and 1 L distilled water) and incubated in the dark at 28 °C for 5 days. Five Pythium-like isolates which were arachnoid-cottony on cornmeal agar were isolated and they all produced hyphal swellings, oogonia, antheridia and oospores. Oospores were globose, smooth and plerotic, with some being aplerotic. The dimensions of hyphal swellings, oogonia and oospores respectively ranged from 9.0 to 21.3 (average 14.1) µm, 12.9 to 22.5 (average 18.2) µm, and 12.5 to 20.5 (average 16.7) µm. Finger-like projections were uniformly distributed on the walls of the oogonia and the antheridia were curved rods. The five Pythium-like isolates were identified as P. spinosum based on morphological characteristics (van der Plaats-Niterink, 1981). Genomic DNA was extracted from the isolates of the Pythium sp. using a DNA extraction kit (OMEGA, U.S.A.). The cytochrome c oxidase subunit I (COI) gene and internal transcribed spacer (ITS) region rDNA were amplified and sequenced using primers FM55/FM52R (Long et al. 2012) and ITS1/ITS4, respectively (White et al.1990). The five COI sequences were aligned and were identical for all five isolates, as well as the five ITS sequences. BLASTn analysis of the 538-bp COI sequence (accession no. MT822775) resulted in a 99% identity with that of the P. spinosum strain CBS122663 (accession no. HQ708832.1), and the 916-bp ITS sequence (accession no. MN847595) showed 100% identity with Genbank accession number AB217665 belonging to P. spinosum. Koch's postulates were confirmed. Corn kernels that had been soaked in water for 24 hours in water, autoclaved for 2 hours at 121˚C and allowed to cool were inoculated with agar plugs of P. spinosum grown on corn meal agar medium (CMA) for 10 days. The inoculated corn kernels were incubated at 28 ℃ for 13～15 days, until the corn kernels were covered with white hypha of P. spinosum. Ten healthy approximately 2-years old American ginseng plants growing in Wengdeng greenhouses were transplanted into a sterilized potting soil that was artificially infested with the corn inoculum (3 g inoculum per 100 g loam mixture). Inoculated and non-inoculated control plants were maintained in a greenhouse with a roof covered with sunshade net at 28 °C and 100% relative humidity. The experiment was repeated once. Four days after inoculation (DAI), the crown of inoculated plants developed water-soaked symptoms similar to those observed in field. No symptoms developed on the control plants. By 7 DAI, the inoculated fine roots and crowns showed water-soaked lesions identical to those observed in field, whereas control plants remained symptomless. The re-isolated isolate of P. spinosum was identical morphologically and by DNA sequence analysis to the original isolate. To our knowledge, this is the first report of root rot on American ginseng caused by P. spinosum in China and worldwide. Identification of the pathogen will assist in devising strategies to protect this important medicine plant from the pathogen, and to prevent yield losses.

Yams (Dioscorea spp.) are widely grown in China as vegetables and for herbal medicine. From 2016 to 2018, water-soaked lesions appeared on the fibrous roots of yam seedlings, and some plants were wilted, up to 15% in Heze city, Shandong Province. Fragments of 1 mm in size were excised from water-soaked roots of diseased plants, dipped in a 0.2% calcium hypochlorite solution for 10 min, placed on a V8 medium, and incubated in dark at 28°C for 5 days. A Pythium sp. was consistently isolated from water-soaked roots of 10 yam plants. The Pythium sp. produced sporangia, oogonia, antheridia, and oospores. Sporangia were terminal or intercalary, filamentous, inflated lobulate. Oogonia were globose, terminal or intercalary, ranging from 20 to 40 µm (average 29.3 µm) in diameter mating with one to five antheridia. Most oospores were globose, smooth, and aplerotic, ranging from 17.8 to 33.5 µm (average 25.3 µm). The antheridia were diclinous and shaped like clavates and crooknecks. Five of the isolates were molecularly identified further. Genomic DNA was extracted from the five isolates of the Pythium sp. using a DNA extraction kit (Omega, U.S.A.). The cytochrome c oxidase subunit I (COI) gene was amplified and sequenced using primers FM55/FM52R (Long et al. 2012). The five COI sequences were determined and identical. BLASTn analysis of COI with a 539-bp sequence (accession no. MH177892) resulted in 100% homology with that of Pythium myriotylum isolate AB292 (accession no. MF374747). Isolates were identified as P. myriotylum based on morphological characteristics (van der Plaats-Niterink 1981) and COI sequences. Koch’s postulates were conducted by first producing inoculum for one of the isolates (SDHZSY-1). Inoculum was produced by first soaking corn kernels for 24 h in water, followed by autoclaving for 2 h at 121°C. Isolate SDHZSY-1 was grown on corn meal agar medium for 10 days, after which agar plugs were transferred to the sterilized corn kernels, which were incubated at 28°C for approximately 15 days, until the corn kernels were covered in white hypha. Ten healthy yam plants were transplanted into a sterilized sandy loam potting soil that was artificially infested with the corn inoculum (3 g of inoculum per 100 g of sandy loam mixture). Inoculated and noninoculated control plants were maintained in a greenhouse at 28°C and 80% relative humidity with a 12-h photoperiod, and the experiment was repeated. All 20 inoculated plants appeared water-soaked within 4 weeks, and P. myriotylum was recovered only from the water-soaked roots of inoculated plants, whereas all 20 control plants remained symptomless in both experiments. To our knowledge, this is the first report of root rot on yams caused by P. myriotylum in China and worldwide. Identification of the pathogen will assist in devising strategies to protect this important plant from the pathogen and to prevent yield losses.

The yam is a perennial twining liana belonging to the family of Dioscorea, and is widely distributed throughout China, where it is planted in open fields. The Chinese yam is rich in nutrition, with positive effects in the prevention of high blood fat and adjustment of the function of the stomach, as well as in the significant reduction of blood sugar, and improvement or adjustment of the immune system. In May 2014, in Heze City, Shandong Province, the stem base of Chinese yam seedlings showed water-soaked lesions, and the fibrous roots also showed hygrophanous lesions. Three Pythium-like strains were isolated from the roots and stems of Chinese yam with disease symptoms, by using general tissue separation on the V8 selective media. The three isolates all were identified as P. ultimum var. ultimum, based on the morphological characteristics and sequence of portions of the cytochrome c oxidase subunit I (CO I) gene. The morphological characterization of colonies on corn meal agar (CMA) revealed colonies of ...

This paper briefly introduced the issues of food safety and environmental pollution caused by pesticide residues in protected vegetables, discussed the status and problems of pesticide use in the protected vegetables in Shandong Province, and analyzed the main factors leading to the pesticide residues, including the low education of most farmers, lack of correct identification of diseases and insect pests, use of pesticides based on personal experience, pesticide preparation by bare hands, large dose of pesticide, frequent application, pesticide spraying without protection, uneven spraying, leakage of pesticide from the sprayers, etc.. Finally, based on the vegetable planting features and advantages in Shandong Province, some suggestions were proposed for references, such as, to enhance the monitoring of pesticide residue, to improve the educational level of farmers and to scentifically use the pesticide.