First report of Pythium aphanidermatum causing crown and root rot on industrial hemp in China

In July 2020, symptoms of crown and root rot were observed on about 10% of 4-month-old plants of industrial hemp Cannabis sativa cultivar Yunma-1 in Weifang City, Shandong Province in eastern China (Fig 1). During this month, the local temperature ranged from 19-32°C, and the total precipitation was 148mm. The disease symptoms included leaf chlorosis, crown and root rot, stunted growth, and wilting (Figs. 1 and 2). The diseased stem and root tissues were collected and cut into fragments of 0.5cm each. The fragments were surface-sterilized by dipping into 1% NaClO for 1 min, rinsed in sterile water and plated on potato dextrose agar (PDA) and on oomycetes-selective medium PARP (Jeffers and Martin 1986). The plates were incubated at 25°C in the dark for 3 days and 18 total single-hyphal purified isolates were obtained for further analyses with 8 from PDA and 10 from PARP. The colonies of all 18 isolates were white, had abundant aerial hyphae, and were cottony in appearance, resembling Pythium spp (Watanabe 2002). The grass-leaf method (Van Der Plaats-Niterink 1981) induced their sexual reproduction. The size and shape of hyphae, oogonia, antheridia, and oospores were all consistent with those of Pythium aphanidermatum (Fig 3). DNA was extracted from three isolates and their internal transcribed spacer (ITS) regions of rDNA were amplified and sequenced using the primers ITS1/ITS4 (White et al. 1990). The ITS sequences of all three isolates were identical to each other (GenBank accession OK091124.1) and showed a 100% query coverage and 99.88% nucleotide sequence identity with that of type strain of P. aphanidermatum (GenBank accession AY598622.2). Pathogenicity tests were performed with three isolates on hemp cultivar B1. Sterile substrates were prepared in 2L-pots containing peat soil and vermiculite in a 2:1 ratio, with test hemp plants grown from rooted stem cuttings. Plants were kept in a greenhouse at 22 to 27°C under 16 h photoperiod, watered every two days (about 200ml each time) and supplied commercial nutrient solution once a week. A month after transplanting to pots, a wound of 1 mm deep and 10 mm long (made by a sterilized needle) on the surface of the root crown area of the main stem was inoculated with an 8-mm-diameter agar disk of mycelia grown on PDA for 4 days. Six plants were tested for each isolate and three plants were inoculated with sterile agar medium without mycelia as negative controls. The experiment was repeated twice. After one month, plants inoculated with P. aphanidermatum isolates showed the same disease symptoms as observed on field plants while all negative control plants remained disease-free. P. aphanidermatum was reisolated from the diseased tissue and confirmed to be identical to those inoculated based on ITS sequencing and colony morphology. To our knowledge, this is the first report of P. aphanidermatum causing crown and root rot on hemp in China. With an estimated 66,700 hectares hemp cultivation in China producing over US$1 billion worth of hemp fiber (McGrath 2020), this pathogen represents a serious threat to the hemp industry. This pathogen has been reported on hemp in the US and Canada (Beckerman et al. 2017; Punja et al. 2018). The origin of P. aphanidermatum on hemp in China and its relationship to those in North America remain to be examined.

تقييم الفعالية البيولوجية للبكتريا المعزولة من داخل النبات في مقاومة مرضي التفحم المغطى وتعفن الجذور وموت البادرات في الحنطة Triticum aestivum L.

أجريت الدراسة لتقييم فعالية بكتريا داخل النبات في السيطرة على المسببات المرضية Pythium aphanidermatum المسبب لمرض تعفن الجذور وموت البادرات و T. caries /Tilletia laevis المسبب لمرض التفحم المغطى، إذ عزلت 50 عزلة من البكتريا من أنسجة نبات الحنطة من بعض مناطق محافظة صلاح الدين. أشارت نتائج إختبار التضاد المباشر للبكتريا المعزولة مع المسبب المرضي P. aphanidermatum إلى أن مدى التثبيط قد تراوح بين (0.3 - 9.3 ملم) إذ بلغ أعلى معدل تثبيط في العزلة SHSt2 بمنطقة تثبيط بلغت 9.3 ملم وشخصت هذه العزلة الى مستوى النوع Pseudomonas flvescens (SHSt2) . أن المعاملة (بذور + رش 1012 خلية بكتيرية/ مل) في التجربة الحقلية قد أبدت تفوقاً معنوياً في مؤشرات النبات الخضرية كمحتوى الكلوروفيل وارتفاع النباتات. أثرت المعاملة ببكتريا P.flvescensفي نسبة الاصابة بالفطرين الممرضين P.aphanidermatum و T. laevis/T. caries إذ بلغت ادنى نسبة اصابة في معاملة البكتريا (بذور + رش 1012 خلية بكتيرية/ مل) بنسبة إصابة بلغت 19.94 و11.13% على التوالي مقارنة باعلى نسبة اصابة في معاملتي السيطرة بوجود الممرضين فقط إذ بلغت نسبة الإصابة 81.57 و 58.47 % على التوالي. في المؤشرات الانتاجية ، رفعت المعاملة بالبكتريا P. flvescens من الوزن الكلي للنبات بوجود الممرضين P. aphanidermatum و T. laevis/T. caries إذ بلغ أعلى وزن 60.4 و 58.71 غم على التوالي في معاملة (بذور + رش 1012 خلية بكتيرية/ مل)، مقارنة بأدنى وزن في معاملتي السيطرة للممرضين إذ بلغ 26.47 و 20.56 غم على التوالي، إضافة إلى ذلك فقد إنعكس تأثير المعاملة بالبكتريا P. flvescens على دليل الحصاد إذ بلغت أعلى نسبة 36.5 و28.75% بوجود الممرضين P. aphanidermatum و T. laevis/T. caries على التوالي في معاملة (بذور + رش 1012 خلية بكتيرية/ مل) مقارنة بأدنى نسبة في معاملة السيطرة للممرضين إذ بلغت 22.42 و18.42 %على التوالي .

Mycogenic Nano-Complex for Plant Growth Promotion and Bio-Control of Pythium aphanidermatum

(1) Background: biological way is one of the most ecofriendly and safe strategies for nanomaterials synthesis. So, biosynthesis-green method was used for the preparation of Zn(II) complex (in the Nano scale) from the reaction of the schiff base ligand 2,2′-((1E,1′E)-(1,2-phenylenebis (azanylylidene)), bis(methanylylidene))bis(4-bromophenol), and Zn(II)sulphate. The biogenic ZnNP-T was characterized by different methods. Our purpose was to evaluate the ability of biosynthesis-green method for the preparation of Zn(II) complex as an antifungal agent against diseases from fungal species. (2) Methods: in this work, isolates of Pythium aphanidermatum and Trichderma harzianum were obtained, and Trichderma harzianum was used to prepare nano metal complex. We tested the pathogenicity of nano metal complex against seedling and germination of seeds, and we evaluated the effectiveness of ZnNP-T for growth promotion of Vicia feba in early stage and inhibitory activity against Pythium aphanidermatum. (3) Results: antagonistic activity of ZnNP-T was tested in vitro against Pythium aphanidermatum, and then the growth rates of Vicia faba were determined. The obtained data revealed that mycelial growth of pathogenic fungus was inhibited about 73.8% at 20 ppm. In addition, improved the total biomass of Vicia faba in the presence of P. aphanidermatum. All concentration of ZnNP-T positively affected root weight of Vicia faba seedlings, and positively affected shoot weight. Root and shoot lengths were affected by using 20 ppm of ZnNP-T with up to 180 and 96.5 mm of shoot and root length compared to that of the control, while germination percentage was significantly enhanced with up to 100% increase after 72 h of germination. (4) Conclusion: one of the modern challenges in vegetable or fruit production is to enhance seed germination and to grow healthy plants with strong root system. In future, there should be a focus on using of biogenic Zinc nano-complex as plant growth promoter agents.

First Report of Pythium aphanidermatum Causing Stalk Rot on Abelmoschus manihot (L. ) Medic in China

Abelmoschus manihot (Linn. ) Medicus (A. manihot) is an annual to perennial herb of the Malvaceae okra, mainly distributed in Guangdong, Guangxi, Fujian, Hunan, Hubei provinces. It can not only be used as an ornamental flower, but also has important economic and medicinal value. Last year, 10% A. manihot in 1,000 acres were observed with stalk rot in the Zhongshang Agricultural Industrial Park, 50 meters east of Provincial Highway 235 in Gaoyang County of Hebei province. Internal discoloration of the stem began brown to black discoloration of the vascular system and became hollow, with the mycelium growing on the surface. Stems from symptomatic plants (approximately 5 mm2) were dissected, washed free of soil, then soaked in 75% ethanol for 16 s to surface-sterilize, and 40 s in HgCl2, then rinsed three times in sterile water. After being dried with blotting paper, five pieces were placed on potato dextrose agar (PDA). After cultured 2 or 3 days, five isolates were purified and re-cultured on PDA in the dark at 25°C. The color of the colony was white. The hyphae were radial in PDA, and the aerial hyphae were flocculent, well-developed with luxuriant branches. The colonies were white and floccus, and the aerial hyphae were well developed, branched and without septum on corn meal agar (CMA). The sporangia were large or petal shaped, composed of irregular hyphae, terminal or intermediate , with the size of (31.6-88.4) μm ×(12.7- 14.6) μm. Vesicles were spherical, terminal or intermediate, ranging from 14.6 to 18.5μm. Oogonia were globose, terminal and smooth which stipe was straight. Antheridia were clavate or baggy and mostly intercalary, sometimes terminal. Oospores were aplerotic, 21.5 to 30.0 μm in diameter, 1.6 to 3.1 μm in wall thickness. The isolates morphological characteristics were consistent with P. aphanidermatum (van der Plaats-Niterink 1981, Wu et al. 2021 ). To identify the isolates, universal primers ITS1/ITS4 (White et al. 1993) were used for polymerase chain reaction–based molecular identification. The amplification region was sequenced by Sangon Biotech (Shanghai, China) and submitted to GenBank (MW819983). BLAST analysis showed that the sequence was 100% identical to Pythium aphanidermatum. Pathogenicity tests were conducted 3 times, with 4 treatments and 2 controls each time. The plants treated were 6 months old. Then the hyphae growing on PDA for 7 days were cut into four pieces. Next, they were inoculated into the soil of the A. manihot. Negative control was inoculated only with PDA for 7 days ( Zhang et al. 2000). The plants were then placed in a greenhouse under 28°C, 90% relative humidity. After inoculated 20 to 30 days, the infected plants showed stalk rot, the same symptoms as observed on the original plants. The control plants didn’t display symptoms. Pythium aphanidermatum was re-isolated from infected stems and showed the same characteristics as described above and was identical in appearance to the isolates used to inoculate the plants. To our knowledge, this is the first report of Pythium aphanidermatum infecting A. manihot stem and causing stalk rot in China. It may become a significant problem for A. manihot. Preliminary management practices are needed for reducing the cost and losses of production.