Prevalence and Molecular Diversity of Plant-Parasitic Nematodes of Yam (Dioscorea spp.) in China, with Focus on Merlinius spp.

There is little information about nematode pests associated with yam in China. Between 2020 and 2021, surveys of yam fields were conducted to investigate the abundance and prevalence of plant-parasitic nematodes in major yam growing areas. A total of 110 bulk soil samples from the yam rhizosphere and 48 yam tubers were collected from seven counties in Jiangxi and Shandong provinces. Standard protocols were used to extract nematodes from soil and tubers and identified at the genus level. In this study, 16 species and 13 nematode genera were recorded. The five most prominent species on the yam rhizosphere according to mean population densities were Pratylenchus coffeae (291/individuals), Meloidogyne (262/individuals), Rotylenchulus reniformis (225/individuals), Merlinius (224/individuals), and Helicotylenchus dihystera (171/individuals). In the tubers, the three most prominent species were Pratylenchus coffeae (415/individuals), Meloidogyne (331/individuals), and Rotylenchulus reniformis (115/individuals). These species were verified with appropriate molecular analysis. The high prevalence of the ectoparasite (Merlinius spp.) on the rhizosphere of yam also revealed that Merlinius spp. May be more important to yam than previously thought. Morphological and molecular analyses further confirmed the identity of the species as Merlinius brevidens and were characterized for the first time on yam in China. Minor morphometrical differences (slightly longer body and stylet) were observed in Chinese populations of M. brevidens compared to the original description. Additionally, this study reveals that M. brevidens isolated from China showed a higher nucleotide sequence in the ITS region compared to M. brevidens populations from India. This finding provides baseline information on the nematode pest occurrence on yam in China and calls for effective management.

Nematicidal activity of cinnamon bark extracts and chitosan against Meloidogyne incognita and Pratylenchus coffeae

Summary In this study, we aimed to evaluate the nematicidal activity of cinnamon bark extracts (CE) and chitosan (Cs) against Meloidogyne incognita and Pratylenchus coffeae under pot and field conditions. In the pot experiments, CE mixed with Cs effectively inhibited M. incognita and P. coffeae infection on robusta coffee plants. The formulations applied, CE:Cs = 8 mg:30 mg, CE:Cs = 16 mg:60 mg and CE:Cs = 16 mg:60 mg per pot, significantly reduced the gall index and nematode number in 5 g of root and 100 g of soil. In addition, the application of CE:Cs = 48 mg:180 mg CE:Cs = 80 mg:300 mg and CE:Cs = 112 mg:420 mg plant−1 effectively reduced root gall formation and nematode density in roots and soil compared with the non-treated control under field conditions. Nematode density in the roots was positively correlated with the rate of yellow leaf disease. These results suggest that cinnamon mixed with chitosan may be used as an effective eco-friendly pesticide against plant-parasitic nematodes.

Occurrence of Pratylenchus coffeae Causing Root Rot of Soybean in Shandong Province of China

Soybean (Glycine max L.) is a very important commercial crop in China (Li et al. 2019). Pratylenchus coffeae (Zimmermann, 1898) Filipjev & Schuurmans Stekhoven, 1941, is one of the most important root-lesion nematodes that invade the roots of many crops. In August 2018, five root and soil samples were collected in a soybean field, near Xipan village in Linshu county of Linyi City, Shandong Province, China (Fig. S1), to investigate the occurrence of root-lesion nematodes. The collected plants (cv. Lindou No.10) were growing poorly and the roots showed distinct brown lesions (Fig. S2). Pratylenchus spp. were extracted using the modified Baermann funnel method for 2 days (Hooper et al. 2005). On average, 395 root-lesion nematodes per kg of soil and 36 root-lesion nematodes per gram of fresh roots were extracted. The extracted root-lesion nematodes were disinfected with 0.3% streptomycin sulfate and cultured on carrot disks for propagation at 25°C. The species identification was based on morphological and molecular criteria. Key morphological features were determined for females and males. Measurements of females (n = 16) included body length = 561.0 μm ± 37.6 (standard deviation) (520.5 to 654.0 μm), tail length = 30.0 μm ± 1.9 (27.0 to 33.5 μm), stylet = 16.0 μm ± 0.6 (15.0 to 17.5 μm), a = 28.2 ± 2.3 (23.7 to 31.5), b = 6.4 ± 0.5 (5.7 to 7.3), c = 18.7 ± 1.8 (15.7 to 23.8), and V = 80.8% ± 2.1 (76.5 to 83.8%). Measurements of males (n = 16): body length = 511.0 μm ± 28.1 (range= 475.5 to 566.0 μm), tail length = 26.0 μm ± 1.3 (23.5 to 28.5 μm), stylet = 15.0 μm ± 0.5 (14.5 to 16.0 μm), spicule length = 17.0 μm ± 0.9 (16.0 to 18.5 μm), a = 30.8 ± 1.5 (28.0 to 33.2), b = 6.1 ±0.4 (5.6 to 6.9), and c = 19.8 ± 1.3 (18.1 to 22.2) (Fig. S3). All the morphological features of this population matched the description of P. coffeae (Castillo and Vovlas, 2007). DNA was extracted from an individual female as described previously (Wang et al. 2011). The rDNA-internal transcribed spacer (ITS) region and the D2/D3 region of the 28S rRNA gene were amplified by primers 18S/26S (Vrain et al. 1992) and D2A/D3B (De Ley et al. 1999), respectively. The PCR products were purified and sequenced. The obtained sequences of the ITS region (1,253 bp) and the D2/D3 region of 28S rRNA (781 bp) were deposited in GenBank. The ITS sequences of the root-lesion nematode obtained in this study (GenBank Accession no. MT879294) exhibited 99% identity with several P. coffeae sequences available in the GenBank (e.g., KR106219, MT586756, KY424205, and MN749379), and the obtained D2/D3 region sequence (MT879295) exhibited 100% identity with several P. coffeae sequences (e.g., MT586754, MN750755, MK829009, and MH730447). Both morphological and molecular data confirmed the presence of P. coffeae. To confirm reproduction on soybean, the obtained root-lesion nematode population was used in a greenhouse (25°C) assay to fulfill modified Koch’s postulates. About 20 days after sowing, eight pots, each with one soybean plant (Lindou No.10) were inoculated with 1000 P. coffeae. The inoculated plants were kept in 1.5 L pots containing 1.2 L sterilized soil. Eight pots of uninoculated soybeans were used as the control. Ten weeks later, the inoculated roots were washed and brown lesions were observed. The number of nematodes/pot was approximately 7360 in soil and 796 in roots, and the reproduction factor was 8.16. Root-lesion nematodes and symptoms were not observed in control groups. P. coffeae has only been reported on soybean in Zhejiang (Wei et al. 2013) and Henan Province (Li et al. 2019) of China. To our knowledge, this is the first report of P. coffeae infecting soybean in Shandong Province, China. Since the root-lesion nematode can cause considerable damage to soybean, care should be taken to prevent the spread of P. coffeae to other regions in China.

Consortium of endophytic bacteria and rhizobacteria effectively suppresses the population of Pratylenchus coffeae and promotes the growth of Robusta coffee

Asyiah IN, Mudakir I, Hoesain M, Pradana AP, Djunaidy A, Sari RF. 2020. Consortium of endophytic bacteria and rhizobacteria effectively suppresses the population of Pratylenchus coffeae and promotes the growth of Robusta coffee. Biodiversitas 21: 4702-4708. Pratylenchus coffeae as the coffee-parasitic nematode can cause a yield loss from 28.7% to 78.4% on Robusta coffee (Coffea canephora A. Froehner). Environmentally friendly and sustainable efforts can be made to control this nematode by applying the antagonistic agents from the bacterial group. The application of antagonistic agents in a consortium has a better effectivity than a single application. This study was aimed to identify the effectivity of the consortium of endophytic bacteria and rhizobacteria formulated on a cost-effective molasse medium against the P. coffeae population and growth of Robusta coffee in a greenhouse. A total of nine bacterial isolates of genus Bacillus and Pseudomonas were used during experiment. The formulation was prepared by mixing the bacterial suspension and 2% molasses with a ratio of 1:1 (v/v). The greenhouse assay was performed following the completely randomized block design pattern with five treatments and five replications. Robusta coffee plant clone BP 936 was treated with a 10 mL (109 CFU mL-1) bacterial consortium mixture while control was treated with 10 mL sterilized water. Each plant was inoculated with P. coffeae 50 J2 after a week of bacterial consortium application. The result showed that all bacterial consortiums were effective in suppressing the population of P. coffeae in soil and root of Robusta coffee. All consortiums gave a better result than control in suppressing the population of P. coffeae. The best suppression of nematodes was shown by the Cst 02 consortium with the suppression value of 62.5% in the root and 76.1% in the coffee plant rhizosphere. The application of bacterial consortium increases the plant height (28.02-44.51%), number of leaves (127.27-168.18%), and leaf area (278.8-425.57%). In this study, suppression of P. coffeae population by bacterial consortium showed a positive correlation with plant growth. This study presents the information of endophytic bacteria and rhizobacteria consortium from Bacillus and Pseudomonas group that is effective in suppressing the population P. coffeae and improves the growth of Robusta coffee.

Fluctuation in Yam Nematodes Depending on the Phenological Stages of the Main Yam Species (Dioscorea alata L.) Cultivated in Côte d’Ivoire

The telluric factors favorable to nematode pathogenic diversity make yam nematode control ineffective. This work aims at studying the fluctuation in yam nematodes depending on yam phenological stages. Trials were implemented in four yam production areas in Côte d'Ivoire. After inventory of symptoms on yam tubers, the nematodes associated with the symptoms were extracted and identified. The correlation coefficients between the severity of symptoms on tubers and the size of the associated nematode populations were determined. The size of nematode populations associated with the symptoms were determined in 100 g of soil and 100 g of yam peel were determined depending on the phenological stages of yam plants. Galls, cracks, dry and wet rot were observed on harvested yam tubers. Globodera, Meloidogyne spp., Pratylenchus coffeae and Xiphinema were the nematodes associated with the symptoms. Pratylenchus coffeae was strongly involved in the development of cracks (r = 0.75) and dry rot (r = 0.86) then Meloidogyne spp. in that of galls (r = 0.78). Pratylenchus coffeae and Meloidogyne spp. fluctuation in cultivation soils and yam tubers is influenced by yam phenological stages. Their numbers increase in soils and tubers before tuberization initiation. Producers could draw on the results of this study to establish a schedule of nematicide treatments that could start as soon as yam seeds are planted.

Tratamento de rizóforos-semente de inhame infectados por Scutellonema bradys e Pratylenchus coffeae com manipueira

RESUMO Sob condições de casa de vegetação foram avaliadas as concentrações de manipueira 25, 50 e 100% e dois períodos de imersão de 9 e 12 h, além da testemunha, no tratamento de rizóforos-semente de inhame naturalmente infectados por Scutellonema bradys e Pratylenchus coffeae. O percentual de brotação dos rizóforos observado aos três meses após o plantio foi de 100% em todos os tratamentos. Aos cinco meses, as concentrações de manipueira nos dois períodos de imersão (25% - 9 horas; 25% e 50% - 12 horas), apresentaram os melhores resultados em relação à testemunha, para as populações finais dos nematoides/g de casca de rizóforos.

Morphological and Molecular Characteristics of Pratylenchus coffeae from the Origin of Robusta Coffee Plantation in Malang, East Java

Pratylenchus coffeae is the most important plant-parasitic nematode in Robusta coffee plantations. Information regarding morphology, morphometric and molecular characters of P. coffeae has not been reported in Indonesia. This study is aimed to describe those characters of P. coffeae that attack Robusta coffee. Root samples were taken from Robusta coffee plantation in Malang, East Java. Nematode extractions was conducted using a mist chamber method. Morphology and morphometric characters were observed from the permanent nematode slides. Single nematode DNA extract was amplified at the D2D3 segment of 28S rRNA and ITS1-5.8S-ITS2 rRNA with universal primers. Amplicon was sequenced and analysed for phylogenetic tree relationships. Female morphological key character of P. coffeae observed are: lip with two annulations, four lateral lines, esophageal overlap with intestine ventrally, monodelphic, and truncated tail shape. Male spicules curved ventrally. Female morphometrics are: n=26, L = 556.4 μm, DGO = 2.4 μm, anterior gonad = 174.8 μm, a = 28.5, b = 6.1, b’ = 4.1, c = 20.1, c’ = 2.3, V = 81.7. A Male is smaller than a female with n=24, L = 505.9 μm, a = 32.3, b = 5.5, b '= 3.9, c = 15.3, c' = 2.8 and T = 40.6. The molecular characters of P. coffeae were investigated for two isolates, namely SA1 and SA2. Based on the D2D3 and ITS1-5.8S-ITS2 regions, isolate SA1 has similarity level of 99% and 97% to the P. coffeae from NCBI. Similar result was shown by Isolate SA2 with similarity of 100% and 100% respectively. Phylogenetic tree analysis using Maximum Likelihood at the D2D3 segment of 28S rRNA and ITS1-5.8S-ITS2 regions showed that P. coffeae in this study was included in one clade with P. coffeae from several countries.

Mobility of Pratylenchus coffeae in segmented soil columns submitted to water flows and plant stimuli

The root-lesion nematode Pratylenchus spp. causes a high-severity disease, promoting high economic losses in various crops of agricultural importance. The nematode control is difficult, especially after introduction into the area. Studies on the migration of this parasite in the soil are scarce. However, understanding nematode behavior in the soil is fundamental to increase the efficiency of management practices, especially in irrigated agriculture. This study aimed to evaluate the influence of water flow and plant stimulus on Pratylenchus coffeae mobility in segmented soil columns. The columns were segmented into 11 rings of 1 cm long filled with washed sand, one of the ends exposed to a bait, and submitted to flows of 0, 3, 7 cm3/min of water. Baits for nematode attraction and repelling consisted of sectioned tubers of yam (Dioscorea cayennensis) and cut leaves of neem (Azadirachta indica), respectively, and the absence of baits as the control. Nematodes were injected into the central segment of the column, and migration and presence in water leaching were determined five days after soil infestation. Mobility of P. coffeae varied with water flow and bait type. In the absence of water flow, nematodes migrated in the opposite direction to the neem baits, but migration was well distributed in the presence of yam baits, with specimens present in all column segments. The nematode had significantly higher resistance to water flow of 3 cm3/min in the presence of yam baits. No nematodes were found in the water leached at flows of 3 and 7 cm3/min in the absence of bait. Key words: Azadirachta indica. Dioscorea cayennensis. Lesion nematode. Soil dispersion.