Fourth-stage Juveniles Research Articles

The function of HgLac in Heterodera glycines and its potential as a control target

The soybean cyst nematode (SCN; Heterodera glycines) is one of the most devastating pathogens for soybean production. The second stage juvenile (J2) invades the host root, develops and form white females which then become brown cysts enter the soil. The brown cyst wall plays a key role in protecting inside eggs from adverse environmental conditions. However, the function of cyst wall tanning (sclerotization and pigmentation) in nematodes is not clear. A browning-related gene discovered from the whole-genome sequencing was cloned and characterized in this study, the gene was confirmed to be the laccase gene and was named HgLac. HgLac mRNA and HgLac protein was detected in the epidermis of juveniles using in situ hybridization and immunolocalization techniques. The HgLac expression level was greater in fourth-stage juveniles (J4s) than in the other stages. Knockdown of HgLac by in vitro RNA interference (RNAi) significantly decreased the infectivity, development and reproduction of J2s but had no effect on cyst wall tanning. Further research revealed that HgLac expression in nematodes was significantly suppressed by 35.41–59.17 % through in planta RNAi, 52.96–58.19 % females could not tan successfully, and the female wall was very soft and fragile, with a low egg hatching rate (1.33 %), which was significantly lower than that of normal females (68.85 %). These results indicate that HgLac plays a key role in cyst wall tanning and suppressing the development and reproduction of the SCN, which provides new ideas for the use of this gene as a target to control SCN.

First report of the maize cyst nematode Heterodera zeae in Sichuan Province of Southwest China.

Maize is the largest crop planted in China. Nine species of cyst nematodes have been reported to affect maize production. Heterodera zeae, H. avenae and Punctodera chalcoensis can cause significant maize yield losses annually (Luc et al. 2005). In 1971, the maize cyst nematode H. zeae was first detected in Rajasthan, India (Koshy et al. 1971). Subsequently, it has been reported in many other countries such as the United States, Greece, Pakistan, and Egypt. In China, H. zeae was first identified in the maize fields of Laibin City, Guangxi Zhuang Autonomous Region (Wu et al., 2017). Cui et al. (2020) identified H. zeae in a maize field of Yuzhou City, Henan Province of Central China in 2018. From 2018 to 2022, a survey of cyst-forming nematodes was conducted in Southwest China. Fifteen soil samples of about 500 g each were collected from Luding County, Ganzi Prefecture of Sichuan Province. No major aboveground symptoms were shown on maize, but a few females were observed on the roots of maize in one field. The cysts and second-stage juveniles (J2s) were collected from each soil sample using Cobb's screening gravity method. A total of 8.50±2.0 cysts per 100 ml of soil on the average were observed in the field. A thin subcrystalline layer was discernible only in young cysts. Morphological and molecular studies of cysts and J2s indicated that the nematodes were identified to be H. zeae in a maize-field. Morphologically, the cysts were in a lemon shape, light brown or pearly white in color. The vulval cone was prominent. Fenestra ambifenestrate, and semifenestra were separated by a fairly wide vulval bridge, fenestral length and width were variable, and the cyst wall was shown in a zigzag pattern. The J2s' body was in a vermiform, tapering at both ends, with a hyaline tail. Stylet was strongly developed with round or slightly anteriorly directed knobs. Morphological measurements of the cysts (n = 9) determined that the mean body length was 417.2 μm (403.6 to 439.4 μm), body width was 429.7 μm (397.6 to 456.9μm); length-width ratio was 1.4 (0.75 to 3); fenestra length was 525.3 μm (498.5 to 570.7 μm); and the mean semifenestra width was 458.6 μm (403.6 to 546.3 μm). Morphometric measurements of second-stage juveniles (n = 20) showed a body length of 419.7μm (355.8 to 492.5 μm); a stylet length of 20.8 μm (19.51 to 23.3μm); a tail length of 41.5 μm (20 to 49.4 μm); and a hyaline tail length of 20.7 μm (16.6 to 24 μm). The main morphological characteristics and measured values were basically consistent with those described by Cui et al. (2022), and all of which were similar to those of H. zeae. Amplification of DNA from random single cysts (n = 5) was conducted using the protocol described by Cui et al. (2022). The rDNA-internal transcribed spacer (ITS) was amplified and sequenced using a pair of universal primers TW81 (5'-GTTTCCGTAGGTGAA CCTGC-3') and AB28 (5'-ATATGCTTAAGTTCAGCGGGT-3'). The ITS sequences were deposited at GenBank with the accession number OR811029.1. Alignments of sequences showed an identity of 98% with H. zeae sequences from China (OP692769.2, MW785772.1) and the USA (GU145616.1), which were confirmed using a pair of species-specific primers HzF1 (5'-GGGGAGGTGAATGTGGG-3') and HzR1 (5'-CCTTTGGCAATCGGTGA-3') of H. zeae with a targeted PCR fragment of 393 bp (Cui et al. 2022). Pathogenicity was conducted and confirmed by infection and reproduction on maize. Seeds (cv. Zhengda 619) were sown in three pots that contained 150 ml of a sterile soil mixture (loamy soil: sand=1:1), and 5 cysts (103 eggs/cyst on the average) were inoculated in each pot at 25/30°C, under a 12-h dark/12-h light condition (Cui et al. 2023). Fifteen days after sowing, third- and fourth-stage juveniles were observed in the rootstained with acid fuchsin, and a total of 32 cysts per maize plant on the average were collected at 40 days after sowing. The new cysts' morphological and molecular characteristics were identical to the cysts from the original soil samples. To the best of our knowledge, this is the first report of H. zeae as a pathogen on maize in Sichuan Province, Southwest China. Our findings will be useful for management and further research of maize cyst nematodes.

First record of cyst nematode (Heterodera filipjevi) on winter wheat from Shanxi Province in North China.

Heterodera avenae, H. filipjevi, and H. laptipons are considered to be the major cyst nematode pathogens affecting most cereals and causing severe crop losses (Smiley and Yan 2015). In China, H. filipjevi was first recorded in Xuchang, Henan Province (Peng et al. 2010). Recently, H. filipjevi has been found in Anhui, Hebei, Shandong and Xinjiang provinces of China (Cui et al. 2021). To further understand the latest occurrence and distribution of H. filipjevi in China, a survey of cyst nematodes was conducted in the wheat planting area of Shanxi Province of North China from June 2018 to November 2020. White female cysts (5.8 ± 2.99 cysts per plant) were found on wheat roots in the sandy soil, and wheat was displaying symptoms of dwarfing, yellowing, and had few tillers in Licheng of Changzhi (N36°32´010´´, E113°27´039´´; N36°29´050´´, E113°23´023´´; N36°29´035´´, E113°22´020´´) and Zezhou of Jincheng (N35°33´057´´, E112°56´020´´) in Shanxi Province, and second-stage juveniles (J2s) were obtained from 13 soil samples using the sieving-decanting method. Four of the 13 samples were identified as H. filipjevi on the basis of morphological and molecular studies of female cysts and J2s. Morphologically, the cysts were lemon shaped and featured a pronounced vulval cone. The color ranged from light to dark brown. The white female shell was covered with a white crystalline layer. The vulval cone was bifenestrate with horseshoe-shaped bullae numerous and distinct, and a strongly developed underbridge. The main measurements (mean ± SD, range) of cysts (n = 13) were as follows: body length including neck 780.5 ± 53.9 μm (692 to 843 μm); body width 527.3 ± 55.5 μm (435 to 620 μm); length/width ratio 1.50 ± 0.21 (1.20 to 1.93); fenestra length 55.5 ± 4.1 μm (49 to 61 μm); fenestra width 24.8 ± 2.2 μm (21.1 to 28.8 μm); vulval slit length 9.0 ± 0.7 μm (7.8 to 9.6 μm); and underbridge length 66.8 ± 5.0 μm (61 to 77 μm). The measurements of J2s (n = 13) were as follows: body length 554.4 ± 23.4 μm (520to 587 μm); stylet length 22.7 ± 0.7 μm (21.5 to 23.8 μm); tail length 61.0 ± 5.5 μm (51.2 to 68.9 μm); and hyaline tail terminus length 37.3 ± 2.7 μm (33.4 to 42.3 μm). These morphological measurements are within the range characteristic of H. filipjevi (Peng et al. 2010). Genomic DNA was extracted from individual cyst (n = 6) and the rDNA internal transcribed spacer (ITS) sequence was amplified using the universal primers TW81 and AB28 (Joyce et al. 1994). The PCR test for each sample was repeated five times. The obtained ITS sequences (GenBank accession No. OQ421499 to OQ421502, 1054 bp) showed more than 99.5% similarity to those of H. filipjevi from the United States (GU079654 and KP878490), Turkey (KR704304 and KR704292), and China (MW789611, KY448473 and KT314234). The results were confirmed again by the species-specific primers HfF1 and HfR1of H. filipjevi and the target PCR fragments of 646 bp were obtained (Peng et al. 2013). The pathogenicity of H. filipjevi was verified by infesting winter wheat (Triticum aestivum 'Wenmai 19') and studying nematode developmentand reproduction with growth chamber (Cui et al. 2015). Eggs were hatched at 14-16°C, and freshly hatched J2s were used to inoculate wheat plants when the roots were approximately 1-centimeter long. Fifteen wheat plants were inoculated with 200 J2s, and three wheat plants without J2s were set as controls (Cui et al. 2021). Parasitic J2s and third- and fourth-stage juveniles were found in roots stained with acid fuchsin at 5, 15, and 25 days after inoculation (DAI), adult females were detected at 50 DAI, and a mean of 23.7 cysts per pot were extracted at 70 DAI (Cui et al. 2015). The morphological and molecular characteristics of the new cysts were identical to those of the H. filipjevi cysts from the original field samples, and no cysts formed in the control groups. Wheat is the main food and economic crop in Shanxi, and H. filipjevi, a potential threat to cereal crop production in Shanxi, should arouse sufficient attention. H. filipjevi is major cyst nematode pathogens of wheat and shows high prevalence in China. The loss of wheat production due to H. filipjevi is as high as 32.3% when the initial density ≥ 64 eggs/mL in soil (Li 2018). To the best of our knowledge, this is the first report of H. filipjevi in Shanxi Province of North China.

Hatching and development of maize cyst nematode Heterodera zeae infecting different plant hosts

The occurrence, distribution, and rapid molecular detection technology of Heterodera zeaeKoshy et al. 1971, have been reported in China. We explored the biological characteristics of H. zeae sampled in Henan Province, China to understand its interaction with plants. Cysts and second-stage juveniles (J2s) were identified under an optical and scanning electron microscope, internal transcribed spacer (ITS) phylogenetic tree, and sequence characterized amplified region (SCAR)-PCR analyses. The optimum hatching temperatures of H. zeae were 30°C and 28°C, with cumulative hatching rates of 16.5 and 16.1%, respectively, at 30 days post-hatching (dph). The hatching rate of H. zeae eggs was improved by 20- and 50-time maize soil leachate and root juice, and 10-time root exudates. The hatching rate in 10-time root exudates was the highest (25.9%). The 10-time root exudates of maize and millet produced the highest hatching rate at 30 dph (25.9 and 22.9%, respectively), followed by wheat (19.9%), barley (18.3%), and rice (17.6%). Heterodera zeae developed faster in maize than in other crops. Fourth-stage juveniles (J4s) were detected in maize roots 8 days post-inoculation (dpi) at 28°C but not in other crops. Combined with hatching tests, the Huang–Huai–Hai summer maize region and the south and central-southwest mountainous maize areas are highly suitable for H. zeae in China. This is the first systematically study of the hatching and infection characteristics on different plant hosts of corn cyst nematode H. zeae in temperate regions. This study laid a theoretical foundation for the rapid spread and high environmental adaptability of corn cyst nematode.

Two Candidate Meloidogyne javanica Effector Genes, MjShKT and MjPUT3: A Functional Investigation of Their Roles in Regulating Nematode Parasitism.

During parasitism, root-knot nematode Meloidogyne spp. inject molecules termed effectors that have multifunctional roles in construction and maintenance of nematode feeding sites. As an outcome of transcriptomic analysis of Meloidogyne javanica, we identified and characterized two differentially expressed genes encoding the predicted proteins MjShKT, carrying a Stichodactyla toxin (ShKT) domain, and MjPUT3, carrying a ground-like domain, both expressed during nematode parasitism of the tomato plant. Fluorescence in-situ hybridization revealed expression of MjShKT and MjPUT3 in the dorsal esophageal glands, suggesting their injection into host cells. MjShKT expression was upregulated during the parasitic life stages, to a maximum at the mature female stage, whereas MjPUT3 expression increased in third- to fourth-stage juveniles. Subcellular in-planta localization of MjShKT and MjPUT3 using a fused fluorescence marker indicated MjShKT co-occurrence with the endoplasmic reticulum, the perinuclear endoplasmatic reticulum, and the Golgi organelle markers, while MjPUT3 localized, to some extent, within the endoplasmatic reticulum and was clearly observed within the nucleoplasm. MjShKT inhibited programmed cell death induced by overexpression of MAPKKKα and Gpa2/RBP-1. Overexpression of MjShKT in tomato hairy roots allowed an increase in nematode reproduction, as indicated by the high number of eggs produced on roots overexpressing MjShKT. Roots overexpressing MjPUT3 were characterized by enhanced root growth, with no effect on nematode development on those roots. Investigation of the two candidate effectors suggested that MjShKT is mainly involved in manipulating the plant effector-triggered immune response toward establishment and maintenance of active feeding sites, whereas MjPUT3 might modulate roots morphology in favor of nematode fitness in the host roots. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Studies on the Growth and Development of Root-Knot Nematode Meloidogyne incognita of Phaseolus vulgaris in Tissue Culture Media

The study was conducted to study the growth and development of root-knot nematode, Meloidogyne incognita of Phaseolus vulgaris in tissue culture media. The data revealed the developmental common bean cv. Paulista of M.S media as the following: the seed swelling stage after three days, the radical appearance stage after five days. The cotyledons leaves appear after six days, the cotyledon leaves appear after eight days, and the seedling stage after twelve days. The life cycle of M. incognita completed about thirty-seven days in root tissues of Paulista cultivar. Microscopic examinations of the infected bean roots revealed the following: Second-stage juveniles of M. incognita penetrated the roots within 24 hrs after nematode inoculation. Third-stage juveniles were observed in roots (six-eight) days after nematode inoculation. While, fourth stage juveniles were observed in roots 12-14 days after nematode inoculation. The pre-adult females were seen inside roots within twenty days after nematode inoculation. The adult females were seen inside roots within twenty-seven days after nematode inoculation. Egg laying females with deposit egg masses of M. incognita was observed thirty-seven days after nematode inoculation.

Postembryonic Development of The Edible Dormouse (Glis Glis Linnaeus, 1766)

The period of postnatal development in the edible dormouse occurs before hibernation, causing a restricted period of growth. Four stages can be identified according to the morphological development and behavior in the nesting period. Dormice are born completely naked, unable to move independently. During the second stage (11–21 days), juveniles acquire a variety of movements. The third stage (22–30 days) begins with opening the eyes and is associated with the activity outside the nest. During the fourth stage (31–44 days) juveniles gradually become independent of their mother. Bright peculiarity of the species postembryonic development is three simultaneous ways of feeding from the 16th day up to the end of lactation on the 39-43rd days: milk, chewed by mother food and firm forages. In social interactions cases of strict aggression are scarce. Dynamic socio-hierarchical structure of litters with the absence of stepped hierarchy is observed. As a whole, during the period of early ontogenesis strategy of the species provides the maximal survival of posterity instead of concurrence inside litters. Geographical differences in time frames of early ontogenesis, found when comparison of data from different regions, maybe caused both by intraspecific variation and peculiarities of local populations.

Life cycle and population dynamics of Bursaphelenchus willibaldi (Nematoda: Rhabditida: Aphelenchoididae) in vitro

Summary To develop models of forest ecosystem functioning, it is necessary to estimate parameters of the population dynamics of nematode species. Parameters of ontogenesis of Bursaphelenchus willibaldi from oak in culture of the fungus Botrytis cinerea were determined in vitro: oviposition began 2.7 (1-3) days after inoculation with adult nematodes; timelines of juvenile formation were: second-stage juveniles: 3.2 (3-4) days, third-stage juveniles: 3.6 (3-5) days, fourth-stage juveniles: 4.8 (3-6) days, first generation (1G) ended with a J4 moulting to adult after 5.8 (4-6) days. One day after the formation of the first generation, nematodes left the 2% potato-sugar agar (PSA) medium with the feed fungus and moved upwards, forming a migratory group (MG). In PSA the total nematode abundance reached peaks on day 11 (2G) and in the MG on day 27 (4.5G). The fecundity of females during population growth was determined as 3.4 (2.1-5.9) eggs day−1 and a model of exponential population growth was developed for total nematodes , ; and for females , . Under conditions of unrestricted reproduction and temperature of 21-23°C, the nematode and fungus can colonise 1 m3 of PSA-like medium in 36.8 days. The survival stage in the propagative generation was presumably J4. The female lifespan consists of a feeding and reproductive phase in the PSA (6 days) and a survival phase in the MG (1 month to 1 year). In MG, nematodes formed swarming groups of 20-200 individuals.

The Meloidogyne graminicola effector MgMO289 targets a novel copper metallochaperone to suppress immunity in rice.

Recent studies have reported that plant-parasitic nematodes facilitate their infection by suppressing plant immunity via effectors, but the inhibitory mechanisms remain poorly understood. This study found that a novel effector MgMO289 is exclusively expressed in the dorsal esophageal gland of Meloidogyne graminicola and is up-regulated at parasitic third-/fourth-stage juveniles. In planta silencing of MgMO289 substantially increased plant resistance to M. graminicola. Moreover, we found that MgMO289 interacts with a new rice copper metallochaperone heavy metal-associated plant protein 04 (OsHPP04), and that rice cytosolic COPPER/ZINC -SUPEROXIDE DISMUTASE 2 (cCu/Zn-SOD2) is the target of OsHPP04. Rice plants overexpressing OsHPP04 or MgMO289 exhibited an increased susceptibility to M. graminicola and a higher Cu/Zn-SOD activity, but lower O2•- content, when compared with wild-type plants. Meanwhile, immune response assays showed that MgMO289 could suppress host innate immunity. These findings reveal a novel pathway for a plant pathogen effector that utilizes the host O2•--scavenging system to eliminate O2•- and suppress plant immunity.

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

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

First record of Heterodera elachista infecting rice in Henan Province of central China.

From June 2018 to November 2019, a survey for cyst-forming nematodes was conducted in rice fields in Henan Province of central China. Cysts were recovered from two rice fields (N32° 14' 048″8 and E115° 4' 008″) at Huangchuan County, leading to more intensive sampling. A further 25 soil samples were then collected with a valve bag from each of these two locations. Cysts and second-stage juveniles (J2) were recovered from roots and soil following Cobb's gravity sieving method. Live cysts were detected in all soil samples with a mean of 6.7±1.5 cysts per 100 ml of soil. Morphologically, the cysts were spherical to lemon-shaped, light to dark brown in color with subcrystalline layer. The vulval cone was well developed, cone terminus with a few large, peripheral, dark brown bullae lacking finger-like projections, and the ambifenestrae were almost rounded with two semifenestrae; width and length of the semifenestrae were similar. The vulval bridge was narrow, with a medium sized underbridge. Cyst measurements (n = 8) determined a mean body length of 431.1 ± 47.23 (351.0 - 516.0) µm, body width 304.3 ± 47.40 (240.0 - 381.0) µm; body length to width ratio 1.42 ± 0.10 (1.2 to 1.6); fenestrae length 39.4 ± 7.06 (26.0 - 47.0) µm; fenestrae width 36.5 ± 5.96 (25.0 - 43.0) µm; vulva slit length 37.1 ± 3.62 (30.0 - 42.0) µm; and the mean underbridge length 75.0 ± 3.39 (70.0 - 81.0) µm. Morphometric J2 measurements (n = 10) included a body length of 432.3 ± 53.26 (379.0 - 512.0) µm; stylet length 20.8 ± 1.87 (18.0 - 24) µm with rounded knobs; tail length 63.1 ± 7.92 (52.0 - 75.0) µm with a hyaline terminal tail length of 35.8 ± 6.14 (28.0 - 45.0) µm. The key morphometrics of this isolate were intermediate to those of the Japanese isolates (Nobbs et al. 1992.) and Chinese isolates (Ding et al. 2012), and other morphological character values were within the range of those reported for Heterodera elachista (Nobbs et al. 1992; Tanha Maafi et al. 2003). Amplification of DNA from single cysts (n = 7) was conducted using the protocol described by Ding et al. (2012). rDNA - ITS sequences were amplified with the universal primers TW81 and AB28 (Joyce et al. 1994). The PCR product was purified and sequenced. The ITS sequences were submitted to GenBank under accession numbers MT579616. Comparisons showed a sequence identity of more than 99.9% for H. elachista sequence MN720080 from Korea and 99.5% for H. elachista sequences JN864884 and JN202916 from China. Species identification was also confirmed using sequence characterized amplified region (SCAR) methods with H. elachista-specific primers He-F/He-R (Qi, 2012). An expected PCR fragment of approximately 434 bp was obtained, which was consistent with those previously reported for H. elachista. Pathogenicity was confirmed by infection and reproduction on rice (Oryza sativa cv. 'Nipponbare'). Seeds were sown into three tubes containing 150 ml of a sterile soil mixture (loamy soil: sand = 1:1), each with 5 cysts (mean of 185 eggs/cyst) and cultivated in an artificial climate box at 25/30°C, under a 12-h dark/12-h light cycle. Three other tubes without cysts were set as control. Two weeks after sowing, stunting and reduction of leaf length were observed and third- and fourth-stage juveniles were observed in roots stained with acid fuchsin. On average, 142 cysts per 150 ml soil were recovered at 5 weeks after sowing. The newly formed cysts corresponded morphologically and molecularly to the cysts from the original soil samples. The globally recognized and economically important rice-damaging cyst nematodes include H. oryzae, H. oryzicola, H. elachista, H. sacchari and H. graminophila (Zhuo et al. 2014). Ohshima (1974) first reported H. elachista, which was originally recorded as H. oryzae in Japan by Luc and Brizuela (1961). H. elachista was then detected from a rice field at Mazandaran Province in Iran (Tanha Maafi et al., 2003), and in upland rice fields in Hunan (Ding et al., 2012) and Guangxi, China (Zhuo et al. 2014). To the best of our knowledge, this is the first report of H. elachista as a pathogen on rice in Henan Province, in central China. According to our field observations, H. elachista was much more serious in direct-seeded rice field than in the transplanted rice fields. H. elachista was also reported attacking corn (Xiao et al., 2019). Henan is the most important corn-producing province in China, thus H. elachista is a potential threat to corn production in Henan. Our findings will be very beneficial for H. elachista management and further research on direct-seeded rice and corn in Henan Province, central China.

Starvation-induced lipid reserve depletion in Pratylenchus brachyurus leads to decreased infectivity in maize roots

Summary Nematode body neutral lipid (triacylglycerol) content has been related to infectivity and has direct implications in control strategies. In this study, Pratylenchus brachyurus populations were split into two groups: i) freshly hatched second-stage juveniles (J2) containing lipids stored during embryogenesis; ii) third- and fourth-stage juveniles (J3/J4) plus females that replenished their lipid reserves by feeding on maize (Zea mays) roots. These groups were subjected to starvation to study their lipid consumption dynamics by staining with Oil Red O, which binds specifically to neutral lipids. Before starvation, freshly hatched J2 had 27% of their body area stained, whereas J3/J4 and females had 75%. Freshly hatched J2 starved for 28 days at 25°C in water lost 63.8% of the original neutral lipid content, which caused a reduction of 91% of infectivity in maize roots. By contrast, J3/J4 and females exposed to the same conditions lost 56.7% of the original neutral lipid content, which resulted in less than 50% reduction in infectivity. During the period of food deprivation, J2 had a mean daily neutral lipid consumption rate of 0.63% and the other infectious stages (J3/J4 and females) had a mean daily neutral lipid consumption rate of 1.46% per day. This study adds information on the dynamics of lipid utilisation that supports the use of longer waiting periods for planting crops after fallow in soils infested with P. brachyurus as compared to Meloidogyne spp.-infested soils.

First Report of Heterodera zeae on Maize (Zea mays) in Henan Province, China

First Report of <i>Heterodera zeae</i> on Maize (<i>Zea mays</i>) in Henan Province, China

Screening of Nematicides against the Lotus Root Nematode, Hirschmanniella diversa Sher (Tylenchida: Pratylenchidae) and the Efficacy of a Selected Nematicide under Lotus Micro-Field Conditions

In Japan, Hirschmanniella diversa is an important pest in lotus cultivation in paddy fields and only lime nitrogen is registered for its control. Therefore, additional nematicides are required to control the nematode. The objective of this study was to screen for an effective nematicide. Fourth-stage juveniles and adults of H. diversa sampled from a lotus field were tested in in vitro solution experiments against 37 pesticides that are registered for the pest control of crops in Japan. Carbamate-based benfuracarb, organophosphate-based fenthion, nereistoxin-based cartap hydrochloride and cyanamide showed nematicidal effects against H. diversa. Benfuracarb at 1 μg/mL showed a nematostatic effect on H. diversa in an agar plate assay. Further, H. diversa treated with benfuracarb did not resume activity 7 days post nematicide treatment when transferred to distilled water. Benfuracarb was tested in micro-field experiments, in which H. diversa density and lotus tuber damage levels were monitored. Results showed that benfuracarb reduced H. diversa densities in the roots during the cultivation period in 2012 and consistently reduced damage levels during a five year study period. Thus, benfuracarb is recommended as an effective nematicide to be used for H. diversa control in lotus cultivation.

Annulohypoxylon sp. FPYF3050 produces volatile organic compounds against the pine wood nematode, Bursaphelenchus xylophilus

Summary Natural volatiles released by the fungus, Annulohypoxylon sp. FPYF3050, were evaluated against the pine wood nematode (PWN), Bursaphelenchus xylophilus. Our results showed that volatile organic compounds (VOCs) caused 64.1 and 58.4% mortality of second-stage juveniles (J2) and mixed-stages (eggs, J2, third- and fourth-stage juveniles, and adults) of populations of PWN, but no inhibitive effects were detected on nematode eggs in the experiment. Analysis of the gases within the Petri plate by gas chromatography-mass spectrometry (GC-MS) showed a yield of an unique volatile with dominant 1,8-cineole in 77.4% relative area (RA) after 72 h treatment of nematodes with Annulohypoxylon sp. FPYF3050 and Botrytis cinerea. The commercial 1,8-cineole at concentrations of 2, 5, 10 and 15 μl ml−1 was applied to examine nematicidal activity. The results showed that 1,8-cineole had a 40-100% inhibition on the nematode eggs during 48 h treatment, more than 82.9% mortality of J2 after 24 h, 48 h and 96 h, and 18.7-91.9% mortality of the mixed-stage population, depending on the period after exposure. This result indicates that 1,8-cineole in the volatile gas emissions of Annulohypoxylon sp. FPYF3050 may play a crucial inhibitory effect on the pine wood nematode The nematicidal volatile gas from fungi may provide a useful biocontrol agent for controlling B. xylophilus.

Marker-assisted selection in Gossypium spp. for Meloidogyne incognita resistance and histopathological characterization of a near immune line

The root-knot nematode, Meloidogyne incognita, is one of the most important parasites that cause economic losses in the cotton crop. Plant genetic resistance is the most desirable strategy to control this pathogen. Sources of resistance in cotton have been known for several years but only a few resistant commercial varieties have been released. Cotton breeding lines were developed using marker-assisted selection in early generation plants to introgress root-knot resistance genes from two different sources: M-315 or CIR1348. Phenotyping was carried out in greenhouse conditions to validate the molecular markers associated with the resistance genes in the breeding lines and confirmed by genotyping. The markers targeting QTLs from M-315 resistance source were highly efficient in the selection of plants resistant to M. incognita, with all plants expressing a reproduction factor inferior to 0.08. CIR1348 resistance source markers were also very efficient in selecting resistance; however, some segregation events revealed the need for fine mapping of the resistance QTLs. To clarify the resistance mechanisms, present in the germplasm derived from the M-315 resistance source, the resistant line CNPA 17-26 B2RF (triple cross [BRS 368RF × M-315] × [BRS 430B2RF]) was chosen for histopathological characterization of plant-nematode interaction and compared with the susceptible FiberMax 966 (FM 966). The second-stage juveniles (J2) penetrated equally in both genotypes. In the histopathological study, a strong blue fluorescence was visualized in the tissues around the nematode (hypersensitivity reaction, HR), mainly at the beginning (from 2 to 6 DAI) in the cortex and central cylinder of the resistant plant, indicating accumulation of phenolic compounds in the roots. At 9 DAI, giant cells in the early stage of subdivision next to nematodes were observed in the central cylinder of the resistant plant, and phenolic compounds were also shown around the nematode. At 12–40 DAI these initial cells were completely degraded with the presence of phenolics involving the nematodes and initial giant cells. No fully developed giant cells or mature females were observed, only fourth-stage juveniles (J4s), and males were frequently visualized at 34 DAI. This resistance mechanism characterizes near-immunity, and so no enlarged females and no egg production were observed. In susceptible control, it was possible to visualize feeding sites well developed from 6 to 30 DAI. Females reached maturity at 26 DAI, and eggs were observed at 30 DAI. Our results suggested that the resistance (near-immunity) of the line CNPA 17-26 B2RF was related to early (2–12 DAI) defense responses that totally prevented nematode reproduction.

Seasonal occurrence and life cycle of lotus root nematode Hirschmanniella diversa (Tylenchida: Pratylenchidae) in lotus roots in paddy fields

The lotus root nematode, Hirschmanniella diversa Sher (Tylenchida: Pratylenchidae), causes damage to lotus tubers, and thus decreases its economic value. Few studies have elucidated the life cycle of H. diversa, which is essential to optimize control methods. Therefore, we collected H. diversa from lotus roots and investigated its seasonal population fluctuations, female mating rates, stylet, and body lengths to understand its seasonal occurrence and possibly predict its life cycle. Hirschmanniella diversa were sampled from two lotus paddy fields between 2010 and 2012 in Ibaraki Prefecture, Japan. The occurrence of H. diversa increased with time peaking around late August and early September, then depreciating thereafter. The seasonal breeding period determined by quantifying the number of H. diversa females with spermatheca filled with sperm peaked from April until mid-May and decreased subsequently. Furthermore, overwintered adult H. diversa females laid eggs around June, hence possibly mating between April and May. Second-stage juveniles emerged at the beginning of August and continued developing thereafter. The fourth-stage juveniles or adults overwintered in the lotus roots, weed roots, or soil. The population dynamics of H. diversa in lotus roots in paddy fields determined in this study will be useful for development of control strategies.

Klebsiella pneumoniae SnebYK Mediates Resistance Against Heterodera glycines and Promotes Soybean Growth.

Soybean is an important economic crop that is often adversely affected by infection in the field with the soybean cyst nematode Heterodera glycines. Biological control is an eco-friendly method used to protect the crop against disease. The bacterium Klebsiella pneumoniae has been reported to protect rice from sheath blight and seedling blight, but its role in the control of nematode is unclear. In this study, the effect of K. pneumoniae SnebYK on the control of H. glycines was assessed. Potting experiment results showed that coating soybean seeds with K. pneumoniae SnebYK not only reduced the infection rate of H. glycines but also decreased the proportion of adult female nematodes. Field experiment results showed that K. pneumoniae SnebYK reduced both the number of H. glycines in soybean roots and the number of adult females. However, K. pneumoniae SnebYK caused low juvenile mortality in an in vitro assay. To further analyze the role of K. pneumoniae SnebYK in the inhibition of H. glycines infection, split root experiments were conducted. The results indicated that K. pneumoniae SnebYK controls H. glycines via induced systemic resistance, which reduces H. glycines penetration. Klebsiella pneumoniae SnebYK treatment also significantly increased the proportion of second-stage juveniles and decreased the proportions of third- and fourth-stage juveniles in the H. glycines population. Moreover, 48 h after inoculation with H. glycines, the expression levels of PR1, PR2, PR5, and PDF1.2 were significantly higher in soybeans pretreated with K. pneumoniae SnebYK than in control soybeans. Interestingly, besides providing protection against nematodes, K. pneumoniae SnebYK fixed nitrogen, produced ammonia, solubilized phosphate, and produced siderophores, leading to well-developed root system and an increase in soybean seedling fresh weight. These results demonstrate for the first time that K. pneumoniae SnebYK not only promotes soybean growth but also inhibits the invasion and development of H. glycines by inducing systemic resistance.

A novel Meloidogyne graminicola effector, MgMO237, interacts with multiple host defence-related proteins to manipulate plant basal immunity and promote parasitism.

Plant-parasitic nematodes can secrete effector proteins into the host tissue to facilitate their parasitism. In this study, we report a novel effector protein, MgMO237, from Meloidogyne graminicola, which is exclusively expressed within the dorsal oesophageal gland cell and markedly up-regulated in parasitic third-/fourth-stage juveniles of M.graminicola. Transient expression of MgMO237 in protoplasts from rice roots showed that MgMO237 was localized in the cytoplasm and nucleus of the host cells. Rice plants overexpressing MgMO237 showed an increased susceptibility to M.graminicola. In contrast, rice plants expressing RNA interference vectors targeting MgMO237 showed an increased resistance to M.graminicola. In addition, yeast two-hybrid and co-immunoprecipitation assays showed that MgMO237 interacted specifically with three rice endogenous proteins, i.e. 1,3-β-glucan synthase component (OsGSC), cysteine-rich repeat secretory protein 55 (OsCRRSP55) and pathogenesis-related BetvI family protein (OsBetvI), which are all related to host defences. Moreover, MgMO237 can suppress host defence responses, including the expression of host defence-related genes, cell wall callose deposition and the burst of reactive oxygen species. These results demonstrate that the effector MgMO237 probably promotes the parasitism of M.graminicola by interacting with multiple host defence-related proteins and suppressing plant basal immunity in the later parasitic stages of nematodes.

Studies on life stages of reniform nematode, Rotylenchulus reniformis on papaya

The study of life cycle consisted of the embryonic and the post embryonic development of reniform nematode. The eggmasses were collected from R. reniformis infested papaya roots and observations were made on embryonic and post embryonic developmental stages. The embryonic development was completed in 3 to 4 days and the eggs hatched from 3rd day and continued upto 7th day. The second stage juveniles remained in water until they developed into fourth stage juveniles or the pre-adults which were formed after 12 to 14 days. The pre-adults are the infective stages of reniform nematode. The pre-adults were then inoculated in papaya seedlings and developed into matured females with laying eggs on 12th day of inoculation. Hence, the total life cycle was found to be 25 to 26 days from egg to egg in papaya.