Detection of Globodera pallida directly from soil sample using mt-COI region based LAMP assay

Potato cyst nematodes (PCN), Globodera rostochiensis (Golden/yellow) and G. pallida (White), are economically important and relatively specialized pest of potato (Solanum tuberosum L.). Both the species are being identified based on cyst colour after 55-60 days after planting (DAP) however, after 65 DAP, we cannot differentiate based on cyst colour as both species turns brown. Moreover, the molecular techniques available to detect the PCN at species level is laborious, time consuming and costly. Therefore, development of rapid, accurate and economically cheap technique for detection of PCN at species level from the field is important to device effective management strategies for sustainable potato production. Accordingly, in the first instance, loop-mediated isothermal amplification (LAMP) assay was developed to detect G. pallida directly from soil by using the mitochondrial (mt-COI) gene specific primer. The LAMP assay was completed within 60 min at 60 °C isothermal conditions and the primer, efficiently detects the G. pallida without any cross reaction with G. rostochiensis, Meloidogyne incognita, M. javanica, Heterodera avenae, H. carotae, and Cactodera spp. In analytical sensitivity tests, the assay was able to detect G. pallida with 1000 times less DNA concentration (10 fg/µl) as compared to conventional PCR (10 pg/µl) and the LAMP product was visualized by using SYBR Gold nucleic acid dye and the assay can be highly useful in detection of G. pallida.

Organic amendments with and without a synthetic nematicide for the management of Heterodera avenae in wheat

Summary This experiment was conducted in the screenhouse of the Department of Nematology, CCS Haryana Agricultural University, Hisar, India, in 2015-2016 and 2016-2017 during the Rabi (winter) season to evaluated organic amendments with and without nematicide for the management of the cereal cyst nematode, Heterodera avenae, in wheat. Organic amendments – castor cake, neem cake, vermi compost and farmyard manure (FYM), individually or with Carbofuran 3G (25 mg pot−1) – were applied in pots 10 days before sowing. All organic amendments significantly reduced the nematode population compared to the inoculated control in both years. The higher doses of the amendments were better at controlling H. avenae than the lower dose. Among the organic amendments, neem cake significantly reduced nematode reproduction compared to other amendments. Significantly fewer cysts, 75 (kg soil)−1, were found in treatment with neem cake at 20 g (kg soil)−1 combined with nematicide. The greatest plant growth parameters were observed under castor cake at 20 g (kg soil)−1 in combination with nematicide. Combined applications of organic amendments with the synthetic nematicide were better than without nematicide.

Wheat root protection from cereal cyst nematode (Heterodera avenae) by fluopyram seed treatment

Cereal cyst nematode (Heterodera avenae), an important plant-parasitic nematode causing yield losses on wheat, has been found in many provinces in China. It is urgent to develop an effective method to protect wheat from H. avenae damage. With a novel mode of action, fluopyram has been registered for controlling root-knot nematodes on cucumber and tomato in China. However, the bioactivity of fluopyram against H. avenae and whether seed treatment is effective in controlling H. avenae on wheat remains unknown. In this study, a bioactivity assay revealed that fluopyram increased the mortality of H. avenae second stage juveniles (J2), with LC50 and LC90 values of 0.92 mg L-1 and 2.92 mg L-1, respectively. Hatching tests showed that H. avenae egg hatching percent decreased 35.2-69.2% with fluopyram at the rate of 1.6-6.4 mg L-1, and the egg hatching period was delayed by 3-9 days compared with the control. In pot and field trials, fluopyram seed treatment significantly reduced H. avenae population density and increased wheat yield by 3.0-13.7%. Therefore, fluopyram seed treatment is an effective approach for the management of H. avenae on wheat in China.

First report of Heterodera filipjevi on winter wheat from Hebei Province in North China

Three of the cereal cyst nematodes, Heterodera avenae, H. filipjevi and H. latipons are considered to be the most economically important cyst nematodes that affect cultivated cereals around the world. H. filipjevi was first detected in China from Xuchang, Henan Province in 2010 (Peng et al. 2010) and now has been recorded in the Central China of Henan, Shandong and Anhui provinces and the Northwest China of Xinjiang Uygur Autonomous Region (Cui et al. 2020). In June 2019, 42 samples consisting of roots and soil were collected from winter wheat fields in Hebei Province of North China. Cysts were detected in 37 soil samples with a mean of 6.4 ± 1.67 cysts per 100 ml of soil. Cysts and second-stage juveniles (J2s) were extracted from root and soil following Cobb's sieving gravity method. Morphological and molecular studies of J2s and cysts confirmed its identity with H. filipjevi in 5 samples from Handan (N36°10'052" and E114°35'056"; N36°37'054" and E114°22'052"), Xingtai (N36°53'060" and E114°30'011") and Shijiazhuang (N 37°26'048" and E 116°05'039") in Hebei Province, China. Morphologically, the cysts are lemon-shaped, light or dark brown in color. The vulval cone is bifenestrate with horseshoe-shaped semifenestrae, strongly globular bullae, and well-developed underbridge. Measurements (mean +_ sd (range)) of cysts (n=10), body length not including neck is 743.0 ± 36.1 μm (665 - 780 μm), body width is 559.0 ± 50.0 μm (455 - 639 μm), length / width ratio is 1.33 ± 0.07 (1.20 - 1.46); neck length is 99.3 ± 8.8 μm (85 - 122 μm); fenestrae length is 56.8 ± 5.0 μm (49 - 65 μm) and width is 25.5 ± 1.8 μm (21.1 - 27.8 μm); underbridge length is 84.0 ± 8.1 μm (62 - 93 μm); and vulval slit length is 8.6 ± 0.5 μm (7.2 - 9.1 μm). Measurements of J2s (n = 12), body length is 541 ± 11.4 μm (490 - 578 μm); stylet length is 22.3 ± 0.5 μm (22.0 - 25.0 μm) with anchor-shaped basal knobs; tail length is 57.7 ± 3.7 μm (52.7 - 65.2 μm), and hyaline tail terminal length is 36.5 ± 2.8 μm (32 - 39.8 μm). The tail had a sharp terminus. Morphology of the cysts and J2s were consistent with the record of H. filipjevi (Peng et al. 2010; Subbotin et al. 2010). The amplifications of rDNA-internal transcribed spacer (ITS) fragments were generated with a PCR fragment of 1054 bp from single cysts of each population, using primers TW81 and AB28 (Joyce et al. 1994). The PCR tests for each sample were repeated five times. The PCR product was purified and sequenced. All nucleotide sequences of ITS-rDNA were submitted to GenBank under accession numbers MW282843-6. Sequences from the ITS region were more than 99.5% identical to those of H. filipjevi from Egypt (KF225725), Turkey (KR704308, KR704293 and MN848333) and China (KT314234, MT254744 and KY448473). These results from ITS supported its identity as H. filipjevi. The results were also confirmed by species specific sequence characterized amplified region primers of H. filipjevi (Peng et al. 2013). Pathogenicity of the H. filipjevi was confirmed by infection of winter wheat (Triticum aestivum L cv. ‘Aikang58’) and examination of the nematode development and reproduction. Wheat seeds were germinated in petri dishes and then transplanted into five polyvinyl chloride tubs (3 cm in diameter, 25 cm in length) that contained 150 cm3 of a sterile soil mixture (loamy soil: sand = 1:1), each with 5 cysts (mean of 252.0 eggs/cyst). Plants were grown in an artificial climate box for one week at 14/18°C, two weeks at 16/20°C, five weeks at 18/25°C and two weeks at 22/30°C, under 8 h of darkness/16 h light and normal culturing practices (Cui et al. 2015). The parasitic J2s, third and fourth-stage juveniles, and adult females were observed in roots stained with acid fuchsin at 10, 20, 30, and 50 days after inoculation (DAI), and an average of 32.0 cysts per tubes were extracted 70 DAI. The new cyst’ morphological and molecular characteristics were identical to the H. filipjevi cysts from the original soil samples. Three other tubes without cysts were set as control and there were no newly formed cysts. Heterodera avenae and H. filipjevi had been detected in a total of 16 wheat-producing provinces in China, which resulted in losses of 1.9 billion CNY year-1 (Cui et al. 2015). To our knowledge, this is the first report of H. filipjevi in Hebei Province of North China. Cereal cyst nematodes are easily transferred to non-infested areas by many avenues, resulting in increased species and pathotype complexity (Cui et al. 2020). Once H. filipjevi continues to spread in main wheat producing area of China, it could become be a new threat to cereals production. It is time to take effective control methods to prevent H. filipjevi further dispersal, especially through the farming machinery transmission. Hebei Province is one of the most important major grain-producing areas, our findings will be very beneficial for H. filipjevi management and further research on winter wheat in Hebei Province, North China.

Cereal cyst nematodes in the western USA.

This chapter focuses on the economic importance, host range, symptoms, damage and biology and life cycle of the cereal cyst nematodes (CCNs) Heterodera avenae and H. filipjevi in the western USA. Information on the interactions between CCN and other nematodes or pathogenic fungi, management strategies and future research and outlook for CCN in the USA are also discussed.

Phytonematodes Associating Wheat in North Eastern Egypt and Pathogenicity of Heterodera avenae on Certain Cereal Cultivars

A survey of plant-parasitic nematodes (PPNs) associating wheat in Ismailia governorate, northeastern Egypt was carried-out during 2016/2017 and 2017/2018 growing seasons. Results showed that seven PPNs genera and/or species were found associating wheat roots. These genera and/or species, in descending order of frequency, were: Tylenchorhynchus spp. (14%), Helicotylenchus spp. (10%), Heterodera avenae (8%), Ditylenchus spp. (5%), Meloidogyne javanica (4%), Pratylenchus spp. and Xiphinema spp. (3%, each). Two screening pot experiments, in two consecutive growing seasons (2017/17 and 2017/18) were carried out to determine the host suitability of 15 Egyptian cereal cultivars including bread wheat (Triticum aestivum), barley (Hordeum vulgare) and oat (Avena sativa) to the cereal cyst nematode, Heterodera avenae, under greenhouse conditions. Results of both experiments were very similar and showed that oat cv. Baladi and wheat cv. Giza 171 were highly susceptible, while wheat cvs. Masr 1, Masr 2, Masr 3, Sakha 95, Seds 1, Seds 12, and Shandawel 1 were susceptible. However, barley cvs. Giza 135, Giza 123, Giza 124, and Giza 125 were found to be moderately resistant to the tested nematode. H. avenae suppressed (P ≤ 0.05) the dry weights of roots, shoots and spikes of the inoculated plants, compared to the non-inoculated checks. Another greenhouse pot experiment was carried out during the wheat-growing season 2018/2019 to determine the effect of different initial population densities (Pi) of H. avenae on the growth parameters of wheat cv. Giza 171 and on nematode reproduction. Results showed that as the nematode Pi increased, both the wheat growth parameters and the nematode reproduction factor (Rf) were decreased (P ≤ 0.05).