Second-stage Juveniles Research Articles

Host-status of sixteen crops used in potato-based crop rotation systems against Meloidogyne javanica in South Africa

Root-knot nematodes (Meloidogyne species) are the most important group of plant nematodes in potato-producing regions of South Africa. Prior to the withdrawal of synthetic chemical fumigant nematicides from the agrochemical markets, Meloidogyne species were viewed as a minor pest in potato-producing regions. Currently, it has been established that all potato cultivars are host to Meloidogyne species, with the preferred management option of the genus being the use of resistant crops. However, the host status of most crops used in potato-based crop rotations intended to manage nematode population densities of the genus had not been established. Globally, M. incognita is viewed as being more aggressive than M. javanica, whereas in South Africa the opposite is true. Therefore, the objective of the study was to determine whether the reproduction potential (RP) values of M. javanica on 16 crops used in potato crop rotation systems would be below unity. The experiment was conducted under greenhouse conditions at the University of Limpopo during autumn (February-April) 2022 and validated in spring (September-November) of the same year in South Africa. Treatments (16 crops) were laid out in a randomized complete block design, with six replications. The crops were inoculated with 250 eggs + second-stage juveniles (J2). At 56 days after inoculation, nematodes variables were collected and expressed as RP, which demonstrated that there were similarities and differences in RP of the 16 crops to infection by M. javanica, but with the values being below one in white maluti oats, tillage radish, forage sorghum, and jap radish. In conclusion, the four crops with RP values below one could be used in a potato-crop rotation system intended to manage the population densities of M. javanica.

Discovery and Mechanism of a Nematicide Candidate (W3): A Novel Amide Compound Containing a Cyclopropyl Moiety.

To find novel nematicides, we screened the nematicidal activity of compounds in our laboratory compound library. Interestingly, the compound N-((1R,2R)-2-(2-fluoro-4-(trifluoromethyl)phenyl)cyclopropyl)-2-(trifluoromethyl)benzamide (W3) showed a broad spectrum and excellent nematicidal activity. The LC50 values of compound W3 against second-stage juveniles of Bursaphelenchus xylophilus (B. xylophilus), Aphelenchoides besseyi, and Ditylenchus destructor are 1.30, 1.63, and 0.72 mg/L, respectively. Nematicidal activities of compound W3 against second-stage juveniles of Meloidogyne incognita were 87.66% at 100 mg/L. Meanwhile, compound W3 can not only observably inhibit the feeding, reproduction, and egg hatching of B. xylophilus but can also effectively promote the oxidative stress adverse reactions of nematodes and cause intestinal damage. Compound W3 can promote the production of MDA and inhibit the activities of defense enzymes SOD and GST in B. xylophilus. Compound W3 can affect the transcription of genes involved in regulating the tricarboxylic acid cycle in nematodes, resulting in weakened nematode respiration and reduced nematode activity and even death. In addition, compound W3 had good inhibitory activity against five pathogenic fungi. Among them, the EC50 of compound W3 against Fusarium graminearum was 8.4 mg/L. In the future, we will devote ourselves to the toxicological and structural optimization research of the candidate nematicide W3.

Efficient and Direct Identification of Ditylenchus destructor and D. dipsaci in Soil and Plant Tissues Using a Species-Specific PCR Assay

Ditylenchus destructor and D. dipsaci are important nematodes that have a significant economic impact on agronomic and horticultural plants worldwide. Microscopic observation alone may not distinguish between D. destructor and D. dipsaci. Accurate and rapid identification of these two species is essential for effective pest management. In the present study, a species-specific PCR assay was developed to detect and differentiate D. destructor and D. dipsaci based on the rDNA-ITS sequences. The primers developed in this study can specifically amplify fragments of DNA from D. destructor and D. dipsaci in the target population, without amplifying DNA from other non-target nematodes within the genus Ditylenchus. The sensitivity test revealed that this procedure has the ability to detect single second-stage juveniles (J2) of D. dipsaci at a dilution of 1/128 and D. destructor at a dilution of 1/64. Additionally, it can detect genomic DNA (gDNA) at concentrations of 10 pg/µL for D. dipsaci and 1 ng/µL for D. destructor. These results align with previously reported results obtained through RPA and LAMP methods. Furthermore, the primers developed in this study for D. destructor not only were able to amplify six different haplotypes of nematodes but also successfully detected it in infested plant roots and soil samples, thereby shortening the time and reducing the number of steps required for detection. Thus, this assay, which does not necessitate taxonomic or morphological expertise, significantly enhances the diagnosis of D. destructor and D. dipsaci in infested fields. This advancement aids in the early control of these nematodes.

Transcriptome Analysis of Meloidogyne javanica and the Role of a C-Type Lectin in Parasitism.

Meloidogyne javanica is one of the most widespread and economically important sedentary endoparasites. In this study, a comparative transcriptome analysis of M. javanica between pre-parasitic second-stage juveniles (Pre-J2) and parasitic juveniles (Par-J3/J4) was conducted. A total of 48,698 unigenes were obtained, of which 18,826 genes showed significant differences in expression (p < 0.05). In the differentially expressed genes (DEGs) from transcriptome data at Par-J3/J4 and Pre-J2, a large number of unigenes were annotated to the C-type lectin (CTL, Mg01965), the cathepsin L-like protease (Mi-cpl-1), the venom allergen-like protein (Mi-mps-1), Map-1 and the cellulase (endo-β-1,4-glucanase). Among seven types of lectins found in the DEGs, there were 10 CTLs. The regulatory roles of Mj-CTL-1, Mj-CTL-2 and Mj-CTL-3 in plant immune responses involved in the parasitism of M. javanica were investigated. The results revealed that Mj-CTL-2 could suppress programmed cell death (PCD) triggered by Gpa2/RBP-1 and inhibit the flg22-stimulated ROS burst. In situ hybridization and developmental expression analyses showed that Mj-CTL-2 was specifically expressed in the subventral gland of M. javanica, and its expression was up-regulated at Pre-J2 of the nematode. In addition, in planta silencing of Mj-CTL-2 substantially increased the plant resistance to M. javanica. Moreover, yeast co-transformation and bimolecular fluorescence complementation assay showed that Mj-CTL-2 specifically interacted with the Solanum lycopersicum catalase, SlCAT2. It was demonstrated that M. javanica could suppress the innate immunity of plants through the peroxide system, thereby promoting parasitism.

Vertical Migration of Second-stage Juveniles of Meloidogyne enterolobii as Influenced by Temperature and Host.

Infective second-stage juveniles (J2) of Meloidogyne spp. migrate towards host roots, which depends on several factors, including root exudates and soil temperature. Although Meloidogyne enterolobii is a highly virulent nematode that affects major agricultural crops worldwide, there is limited ecological data about it. The objective of this study was to determine the J2 migration pattern vertically in 14-cm long segmented soil columns towards tomato (Solanum lycopersicum) and marigold (Tagetes patula) roots, each grown at two soil temperatures (20 or 26ºC). Bottomless cups with tomatoes or marigolds were attached to the top of each column; cups with no plants were used as untreated controls. Juveniles (1,000/column) were injected into a hole located 1 cm from the bottom of each column. The apparatuses were placed in growth chambers at 20 or 26ºC, and J2 were allowed to migrate for 3, 6, 9, or 12 days after injection (DAI). At each harvest, J2 were extracted from each ring of the columns and counted to compare their distribution, and root systems were stained to observe root penetration. M. enterolobii migrated over 13 cm vertically 3 DAI regardless of temperature, even without plant stimuli. The vertical migration was greater at 26ºC, where 60% of active J2 were found at distances >13 cm at 12 DAI. Temperature did not affect root penetration. Overall, a greater number of J2 was observed in tomato roots, and root penetration increased over time.

First Report of Root-knot Nematode, Meloidogyne enterolobii on Passiflora edulis in Yulin, China.

Passion fruit (Passiflora edulis), a medicinal plant, was introduced into China in the early 19th century, is mainly cultivated in southern provinces (Liang et al. 2019). During March 2023, a survey was carried out and 167 samples were taken from passion fruit cultivated area in Yulin (22.6570263°E; 110.1765019°N) apart from the planting base appeared yellow leaves, stunted growth, and distinctive galls on the roots. Within the galls, Meloidogyne sp. females and egg masses were observed. From the rhizosphere soil, second-stage juveniles (J2) were extracted, and population density was 105/500 g soil. The species was determined to be Meloidogyne enterolobii based on morphological characteristics, including female perineal pattern, and genetic analyses. Female (n = 10) perineal patterns showed oval shape, with coarse and smooth striae, dorsal arch rounded to square, and lateral lines not distinct. The male head cap was high and rounded, with the head region only slightly set off from the body, knobs large, ovoid to rounded. The measurements of males (n = 10) included body length, 1,230.7 ± 244.94 (997 to 1,569) µm; a, 38.58 ± 7.8 (33.45 to 47.05) µm; c, 113.03 ± 26.22 (80.82 to 144.23) µm; stylet, 15.68 ± 1.1 (14.5 to 17.4) µm; spicules, 31.83 ± 2.84 (28.69 to 36.1) µm; tail, 11.09 ± 1.72 (8.02 to 13.38) µm; and gubernaculum length, 8.34 ± 0.28 (8.11 to 8.98) µm. Measurements of J2 (n = 20) included body length, 455.75 ± 44.94 (381 to 512) µm; a, 26.32 ± 3.89 (18.18 to 32.70) µm; c, 8.56 ± 1.2 (6.36 to 10.80) µm; stylet, 12.44 ± 0.76 (11.2 to 13.8) µm; DGO, 3.65 ± 0.54 (2.84 to 4.68) µm; tail, 53.89 ± 6.36 (39.8 to 62.2) µm; and hyaline tail terminus, 11.77 ± 2.83 (7.14 to 16.2) µm. These morphological characteristics are similar to those reported in the original description of M. enterolobii (Yang and Eisenback 1983). The sequences of the partial ITS region was amplified with V5367 (5'-TTGATTACGTCCCTGCCCTTT-3') and 26S (5'-TTTCACTCGCCGTTACTAAGG-3') primers (Vrain et al. 1992). The region between cytochrome oxidase subunit II (COII) and the 16S rRNA mitochondrial DNA (mtDNA COII) was also amplified with the primers C2F3 (5'-GGTCAATGTTCAGAAATTTGTGG-3') (Powers and Harris 1993) and MRH106 (5'-AATTTCTAAAGACTTTTCTTAGT-3') (Stanton et al. 1997). The ITS region yielded a fragment of 757 bp (OR072957) and mtDNA COII of 706 bp (OR078415). A BLAST search indicated the sequences were 100% identical to several sequences of M. enterolobii (MT406250, MH756127 and AY831967, MN269940, respectively). To confirm pathogenicity, 20 passion fruit (P. edulis Sim. f. flavicarpa) 30-day-old seedlings were transplanted into pots with an autoclaved mixture of sand and field soil (3:1) and maintained in the glasshouse at 25 ± 2°C with 65 ± 5% relative humidity. After eight weeks, fifteen plants were inoculated with 500 J2/pot (nematode culture collected from the original field), and another five uninoculated plants served as a control. Two months later, aboveground symptoms were similar to those observed in the field. Nematode reproduction occurred and root galls were observed. The reproduction factor (nematode final population density/initial population density) was 4.8. The disease caused by M. enterolobii was severe in Yulin city of Guangxi. Guangxi is an important area for passion fruit culture, with about 2000 ha, which is responsible for two-thirds of China production (Xing et al. 2020). This is the first record of P. edulis natural infection with M. enterolobii in the Yulin City of Guangxi, China.

First Report of Meloidogyne enterolobii Infecting Yellow pitaya (Selenicereus megalanthus) in China.

Yellow pitaya, Selenicereus megalanthus, is a night-blooming, climbing cacti of tropical origin, which has received increasing attention for its potential as a new exotic fruit crop (Lichtenzveig et al. 2000). The crop is grown extensively in Hainan Province, China (3000 ha). In October 2021, a survey was conducted on a farm located in Changjiang (19°21'4″N, 108°47'2″S), Hainan Province, China. Some yellow pitaya plants were found that were stunted and chlorotic, with abnormally thin stems (Fig. 1B), and no symptoms on healthy plants (Fig. 1A). Dead plants were also observed. Many galls and females with egg masses were observed on roots (Figs. 1C & 1D). This is typical of root-knot nematode (RKN) infections, and the incidence of infection was 36.7%. Meloidogyne sp. females and egg masses were dissected from roots of the infected plants. The perineal pattern of females (n= 5) was round to oval-shaped with a high dorsal arch (Figs. 1I & 1J). Second-stage juveniles (J2s) had truncated lips (Figs. 1E & 1F) and long-conical tails with bluntly rounded tips (Figs. 1G & 1H). The J2s body length (n= 24) averaged 416.79 μm (349.21 to 472.76 μm) with a mean width of 15.36 μm (12.47 to 17.52 μm); mean stylet length was 11.16 μm (10.10 to 13.23 μm); tail length averaged 53.73 μm (43.46 to 65.90 μm). The morphological characteristics matched the original description of M. enterolobii (Yang and Eisenback 1983). Males were not found. Genomic DNA was extracted from eight single J2s, and the mitochondrial (mtDNA) region between COII and 16S rRNA gene was amplified with primers C2F3/1108 (Powers and Harris 1993). A 652-bp DNA fragment was obtained, for which the sequence (GenBank accession no. OP122499) was 100% identical to the sequences of M. enterolobii isolates from China（MN269947）and the USA (MN809527). Furthermore, species identification was also confirmed using M. enterolobii specific primers Me-F/Me-R. An amplicon size of ∼230 bp was obtained, which is consistent with those previously reported for M. enterolobii (Fig. 2) (Long et al. 2006). Therefore, this population was identified as M. enterolobii based on morphological and molecular characteristics. Pathogenicity tests were performed in the greenhouse at 26℃ and 80% relative humidity with a 14-h/10-h light/dark photoperiod. Ten RKN-free S. megalanthus seedlings were transplanted into pots containing sterilized soil. After 3 weeks, the roots of 5 plants were inoculated with 3,000 eggs and J2s of M. enterolobii per plant. Five uninoculated plants were used as control plants. After 2 months, no galling or symptoms were observed on the control plants. All inoculated plants had galled roots similar to those observed in the field. Females and egg masses were obtained by dissecting galls. The nematode reproduction factor (RF= final population/initial population) was 1.9. Adult females (n= 5) dissected from inoculated plants were identified as M. enterolobii with sequence-specific primers Me-F/Me-R, thus confirming pathogenicity. The pathogenicity test was carried out twice with similar results. M. enterolobii is one of the most damaging species of RKN, due to its wide host range, high level of pathogenicity, and ability to develop and reproduce on several crops with resistance genes to other RKN (Castagnone-Sereno 2012). To our knowledge, this is the first report of S. megalanthus (yellow pitaya) as a host of M. enterolobii in China. Further studies are needed to develop and evaluate integrated management strategies.

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 report of Meloidogyne hapla on hemp (Cannabis sativa) in Oregon.

Hemp is a crop that has gained interest in Washington and Oregon. As with other crops, hemp production faces challenges due to biotic factors, including plant-parasitic nematodes. During a survey for plant-parasitic nematodes associated with hemp, Meloidogyne sp. was found in a composite root sample collected in Oregon. Morphological characterization of second-stage juveniles identified the nematode as Meloidogyne hapla. Molecular identification confirmed the population as M. hapla. To our knowledge, this is the first report of M. hapla on hemp in the Pacific Northwest of the United States.

Characterization and Effect of a Nematophagous Fungus Talaromyces cystophila sp. nov. for the Biological Control of Corn Cyst Nematode.

The dynamic of plant-parasitic nematode populations in soil is closely related to soil microorganisms. Fungi from Heterodera zeae cysts were isolated to explore the phenomenon of decline in the H. zeae population in the soil. Phylogenetic study of partial ITS, BenA, CaM, and RPB2 gene sequences, in addition to morphological investigations, was utilized to identify a nematode-destroyingfungus. The nematicidal activity of a novel strain GX1 against H. zeae was assessed in vitro and in the greenhouse. Our findings revealed that strain GX1 is a new species of Talaromyces, named Talaromyces cystophila. It has a strong parasitic and lethal effect on H. zeae cysts, with 91.11% parasitism on cysts at 3 days after treatment. The contents of second-stage juveniles (J2s) and eggs inside the cysts were degraded and formed dense vacuoles, and the damaged eggs could not hatch normally. The spore suspension exhibited high nematophagous activity against nematodes, and fermentation filtrate exhibited marked inhibition of egg hatching and nematicidal activities on J2s. The hatching inhibition rates of eggs exposed to 1 × 108 CFU/ml spore suspensions or 20% 1-week fermentation filtrate (1-WF) for 15 days were 98.56 and 100%, respectively. The mortality of J2s exposed to 1 × 108 CFU/ml spore suspension reached 100% at 24 h; exposure to 50% 2-WF was 98.65 and 100% at 24 and 48 h, respectively. Greenhouse experiments revealed that the spore suspension and fermentation broth considerably decreased H. zeae reproduction by 56.17 to 78.76%. T. cystophila is a potential biocontrol strain with nematophagous and nematicidal activity that deserves attention and application.

Evaluation of nematicides for Meloidogyne enterolobii management in sweetpotato.

Sweetpotato is an important crop whose roots are consumed by people worldwide. Meloidogyne enterolobii stands out as a highly deleterious variant among the species of root-knot nematode that causes significant damage in sweetpotato. In the present study, the activity of four nematicides against M. enterolobii was assessed both in vitro and in growth cabinet experiments. After 48 hours of exposure, fluopyram and cyclobutrifluram had a greater negative effect on the motility of M. enterolobii second-stage juveniles (J2s) compared to fluensulfone and hymexazol, with respective median effective concentration (EC50) values of 0.204, 0.423, 22.335 and 216.622 mg L-1. When M. enterolobii eggs were incubated for 72 hours at the highest concentration of each nematicides, the inhibitory hatching effect of cyclobutrifluram (2.5 mg L-1), fluopyram (1.25 mg L-1) and fluensulfone (80 mg L-1) surpassed 85%, whereas hymexazol (640 mg L-1) was only 67%. Similar results were observed in growth cabinet experiments as well. The disease index (DI) and gall index (GI) were significantly decreased by all four nematicides compared to the control. However, the application of hymexazol did not yield a statistically significant difference in the egg masses index compared to the control, a finding which may be attributed to its potentially limited penetrability through the eggshell barrier. Overall, this study has demonstrated that all four nematicides effectively suppress M. enterolobii in sweetpotato, and this is the first report on the nematicidal activity of cyclobutrifluram and hymexazol against M. enterolobii.

Identification and Biological Characterization of a New Cyst Nematode, Heterodera glycines sbsp.n. tabacum, Parasitizing Tobacco in China.

In an investigation of diseases from plant-parasitizing nematodes in Henan Province, a cyst nematode was found on tobacco roots and in rhizosphere soil. We identified this strain as a new cyst nematode subspecies, Heterodera glycines sbsp.n. tabacum. The cysts and second-stage juveniles (J2s) parasitizing Henan tobacco were larger than those of H. glycines. A single 345-bp fragment was amplified from H. glycines sbsp.n. tabacum, whereas the 345- and 181-bp fragments were amplified from the soybean cyst nematode. Thus, H. glycines sbsp.n. tabacum was distinct from H. glycines. There were base transversions at 504 sites and base transitions at 560, 858, 920, and 921 sites in the rDNA-ITS sequences of H. glycines sbsp.n. tabacum compared with H. glycines, and there were base transitions at 41, 275, 278, and 380 sites in the mtDNA-COI sequences. In the phylogenetic tree based on the rDNA-ITS and mtDNA-COI regions, H. glycines sbsp.n. tabacum was clustered on a single branch. Based on the randomly amplified polymorphic DNA (RAPD) technique, sequence characterized amplified region (SCAR)-PCR primers were designed. A single 1,113-bp fragment was amplified by specific primers (HtF1/HtR1) from H. glycines sbsp.n. tabacum, while no fragments were obtained from H. glycines. The H. glycines sbsp.n. tabacum can infect soybean plants but cannot complete its life cycle on soybean. Eleven tested tobacco cultivars were infected, with an average reproduction factor (Rf) of 9.74 and a maximum of 64.2 in 'K326'. The cumulative egg hatching rate of H. glycines sbsp.n. tabacum in the presence of tobacco root exudates was 42.6% at 32 days posthatching, which was significantly greater than that in the presence of soybean root exudates (30.3%) or sterile water (33.1%). In summary, the cyst nematode population parasitizing Henan tobacco was identified as a new subspecies, H. glycines sbsp.n. tabacum.

Host Suitability of Summer Cover Crops and Peach Rootstocks to the Peach Root-Knot Nematode, Meloidogyne floridensis.

Greenhouse experiments were conducted to determine the host suitability of ten summer cover crops and two peach rootstocks to Meloidogyne floridensis by inoculating them with 10,000 M. floridensis eggs. Brown top millet and sunn hemp were nonhosts as they did not support nematode reproduction. Buckwheat, cowpea, pearl millet, Japanese millet, and sunflower supported more than 25,000 eggs/pot, which indicated that these crops are good hosts to M. floridensis. The crops that supported poor nematode reproduction were sesame, grain sorghum, and sorghum-sudangrass, with their reproduction ranging from 219 to 7,750 eggs/pot. In addition to having many galls on the roots, the peach rootstock Guardian had 10,100 eggs on the roots and 450 second-stage juveniles in the pot, which indicated that 'Guardian' is a good host to M. floridensis. Although the nematode reproduction on MP-29 rootstock was relatively lower, the presence of many large galls on the roots indicates MP-29 is susceptible to M. floridensis. Results from the current study suggest that the employment of nonhost cover crops and poor-host rootstocks could aid in effective nematode management programs for peaches.

Eryngium foetidum Reported as a New Host of Meloidogyne enterolobii in the State of Pará, in the Eastern Amazon Region, Brazil

Amazon chicory (Eryngium foetidum L. [Apiaceae]), also known as culantro, is native to Tropical America and the West Indies. It belongs to the unconventional food plants (UFPs) group, and in addition to be consumed as a spice herb, it possesses a wide range of ethnomedicinal uses (Paul et al. 2011). In 2019, in the eastern Amazon region of Brazil, state of Pará, producers of E. foetidum in the municipality of Castanhal (01°15'363” S 047°10'232” W) reported the occurrence of underdeveloped plants with leaf yellowing and a large number of galls in the root system, which are typical symptoms of root-knotting nematode. Soil and root samples were collected and sent to the Nematology Laboratory (LabNema) located at the Faculty of Agrarian and Veterinary Sciences, UNESP, Jaboticabal, São Paulo, Brazil. A total of 46 second-stage juveniles (J2s) were extracted per 100 cm3 of soil, and a total of 460 eggs and J2s Meloidogyne spp. were found per gram of root. Morphological and molecular techniques were used to identify the species. The analysis of the perineal patter of ten females revealed thin striations in an oval shape with a high and semi-trapezoidal dorsal arch. No striations were observed in the perivulvar region. The labial region of the ten males analyzed exhibited a non-prominent labial disc, fused and slightly recessed submedian lips, with no apparent annulations. The morphological characteristics observed in the adults were consistent with those originally described for Meloidogyne enterolobii (Yang; Eisenback, 1983), confirming the species purity of the recovered population. Three individual nematodes had their 18S rDNA region sequenced (Holterman et al. 2006) which showed an average identity of 99.7% with other sequences of M. enterolobii available in the GenBank database. A Bayesian phylogenetic tree was constructed, providing insights into the specific relationship of M. enterolobii recovered from E. foetidum with other related nematodes. Each of the three sequenced nematodes represented a unique haplotype, resulting in their separation into distinct clades. Moreover, the obtained sequences presented polymorphisms that differed from the M. enterolobii sequences already available in the database, highlighting the genetic diversity of this species in relation to its original host (Silva et al. 2021). The species M. enterolobii was also confirmed using species-specific primers for M. incognita, M. javanica, and M. enterolobii (Zijlstra et al. 2000; Tigano et al. 2010). To confirm the pathogenicity of M. enterolobii on E. foetidum, a modified Koch Postulate was conducted. Six seedlings of E. foetidum were transplanted individually to 10-liter pots containing autoclaved soil. Each pot was then inoculated with 5 mL of a suspension containing 3,000 eggs and J2s from the original population of M. enterolobii obtained from E. foetidum. After 90 days, the inoculated plants exhibited root galls with a plentiful egg mass, in contrast to the healthy non-inoculated plants. The average number of M. enterolobii nematodes recovered from the roots of the inoculated plants was 42,040 eggs and J2s, resulting in a reproduction factor (RF) of 14.0. The importance of reporting the occurrence of M. enterolobii in E. foetidum is due to the fact that this plant species is cultivated in a crop rotation system with other vegetables such as lettuce and coriander, which are also hosts of M. enterolobii. Consequently, different crop rotation strategies and control alternatives need to be considered in areas where E. foetidum is grown. This is the first report of E. foetidum serving as a host for the root-knot nematode M. enterolobii worldwide.

First Report of Root-Knot Nematode, Meloidogyne javanica, Infecting Stachys byzantina in São Paulo, Brazil

Stachys byzantina belongs to the Labiatae and is known by the names "peixinho-da-horta" (Brazil) and "lamb's ear" (USA). Its importance is associated with its medicinal properties (Bahadori et al. 2020) and nutritional aspects (Milião et al. 2022). Root-knot nematodes cause severe damage to plants and suppress production. In January 2021, plants of S. byzantina in the municipality of Jaboticabal (21°14'38.7"S, 48°17'10.6"W) showed symptoms of reduced growth, yellowed leaves and the presence of galls in the roots. Initially, samples of roots from a S. byzantina were analyzed at the Nematology Laboratory (LabNema/UNESP), Jaboticabal, Brazil, estimating 20,000 eggs and juveniles of Meloidogyne sp. in 10 g of roots. To confirm the host ability of the species, a pathogenicity test was performed using Koch's postulate. For this purpose, the test was conducted in a greenhouse where 3,000 eggs and second-stage juveniles (J2) were inoculated onto three plants (n=3) of S. byzantina. After 90 days, the inoculated plants showed the same symptoms as those observed in the field. No symptom or nematode was detected in the uninoculated plant (control). Nematodes were extracted from the roots of inoculated plants and quantified. The perineal pattern of females (n=10) (Netscher and Taylor, 1974) and the labial region of males (n=10) (Eisenback and Hirschmann, 1981) were analyzed and compared with the morphological characteristics of the original description of the species (Chitwood, 1949). For analysis based on esterase isozyme phenotype, the α-method of Esbenshade and Triantaphyllou (1990) was used, and females (n=7) were examined. To confirm identification, whole genomic DNA from an adult female (n=1) was extracted using the Qiagen DNeasy® Blood &amp; Tissue Kit and this sample was used for both genetic sequencing and the sequence-characterized amplified region techniques (SCAR). PCR amplifications were performed for the 18s rRNA gene using primers 988F and 1912R from Holterman et al (2006). Our sequence was deposited in GenBank (NCBI) under the identifier OP422209. Finally, species-specific SCAR primers (Fjav/Rjav, Me-F/Me-R, and Finc-F/Finc-R) designed by Zijlstra (2000) were used to identify Meloidogyne spp. Koch's postulate analysis yielded the following results: (n=1) 9,280 eggs and J2 (Reproduction factor, RF = 33.09); (n=2) 111,720 eggs and J2 (RF = 37.24); (n=3) 59,700 eggs and J2 (RF = 19.9) (RF mean = 30.08). The following characteristics were observed in the perineal region of females: Low and rounded trapezoidal dorsal arch with two distinct lateral lines clearly separating the dorsal and ventral arch regions, similar to the morphological features of the species description by Chitwood (1949). Males had a convex labial plate with a non-raised labial disk joining the submedial labia, a non-rugged labial region, the basal tubercles were usually wider than high, and a rounded tail tip (Eisenback and Hirschmann 1981). The α-esterase enzyme profile showed the J3 phenotype typical of M. javanica (Rm [×100] = 46.0, 54.5, and 58.9). The 18s rRNA sequences grouped Meloidogyne sp. with species such as M. enterolobii, M. incognita, and M. javanica. A DNA fragment of about 700 bp was amplified with Mj (Fjav/Rjav) primers, but not with Me (Me-F/Me-R) and Mi (Finc-F/Finc-R) primers, which confirmed the identification of M. javanica. Accurate identification and characterization of the occurrence of new hosts of M. javanica will allow us to determine the range and geographic distribution of the species. This is the first report on the occurrence of M. javanica on S. byzantina in Brazil. This report is important so that management strategies can be applied to prevent the spread of the pest to other areas.

Biocontrol Efficacy and Induced Resistance of Paenibacillus polymyxa J2-4 Against Meloidogyne incognita Infection in Cucumber.

Meloidogyne incognita is one of the most destructive agricultural pathogens around the world, resulting in severe damage to yield and quality in agricultural production. Biological control promises to be a great potential alternative to chemical agents against M. incognita. Paenibacillus polymyxa J2-4, isolated from ginger plants injured by M. incognita, has shown excellent biocontrol efficacy against M. incognita in cucumber. In vitro experiments with the strain J2-4 resulted in a correct mortality rate of 88.79% (24 h) and 98.57% (48 h) for second-stage juveniles (J2s) of M.incognita. Strain J2-4 significantly suppressed nematode infection on potted plants, with a 65.94% reduction in galls and a 51.64% reduction in eggs compared with the control. The split-root assay demonstrated that strain J2-4 not only reduced J2s' invasion but also inhibited nematode development through the dependence on salicylic acid and jasmonic acid signaling of strain J2-4 induction of plant resistance in local and systemic roots of cucumbers. Genomic analysis of strain J2-4 indicated biosynthetic gene clusters encoding polymyxin, fusaricidin B, paenilan, and tridecaptin. In addition, genetic analysis showed that none of the genes encoding virulence factors were detected in the genome of J2-4 compared with the pathogenic Bacillus species. Taking all the data together, we conclude that P. polymyxa J2-4 has potential as a biological control agent against M. incognita on cucumbers and can be considered biologically safe when used in agriculture.

Differences of Pine Wood Nematode (Bursaphelenchus xylophilus) Developmental Stages under High-Osmotic-Pressure Stress.

Under ion imbalance, water deficiency, and salt stress, the osmotic pressure of the tree sap increases, and pine wood nematodes (Bursaphelenchus xylophilus, PWN) parasitizing in the trees may be subjected to high-osmotic-pressure stress. KCl, L-malic acid, sucrose, and glycerol solutions were used as osmolytes to explore the highest osmotic concentration that PWN can tolerate. Survival analysis showed that when the treatment concentration exceeded 90%, only a few nematodes in the glycerol group survived under 6 h treatment, and most of the survivors were third-stage dispersal juveniles (DJ3). Further examination revealed that under different concentrations of glycerol-induced high osmotic pressure, the survival rate and body length change rate were the highest in the DJ3 and the lowest in the second-stage propagative juveniles. In order to explore the molecular mechanism of resistance of DJ3 to high osmotic stress, transcriptome sequencing was performed at each developmental stage of PWN and differentially expressed genes that were up-regulated or down-regulated only in DJ3 were screened. The expression of genes related to CoA in DJ3, a key enzyme in metabolism, was significantly higher than the other developmental stages. In addition, the expression of the anti-reversal signal pathway-related gene AKT-1 in DJ3 was significantly lower than in the other development stages. Therefore, the specific expression of genes in DJ3 under high osmotic pressure may help them rapidly produce and accumulate energy-related compounds and activate the adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) pathway to respond to damage caused by high-osmotic-pressure stress in time, thus promoting survival.

Reaction of Brazilian genotypes of pulses (pea, chickpea and lentil) to the root-knot nematode Meloidogyne enterolobii

Summary The root-knot nematode, Meloidogyne enterolobii, is an important pathogen of numerous crops, including the so-called pulses. Hence, it is necessary to identify genetic resistance, as it is the most efficient, cost-effective, and environmentally sound way to manage nematodes in the field. The objective of this study was to screen a Brazilian germplasm collection of pulse crops (peas, chickpeas, and lentils) comprising accessions from the Embrapa Germplasm Bank and commercial cultivars against M. enterolobii under glasshouse conditions. The experiment was conducted with 23 treatments (genotypes), i.e., 14 pea, six chickpea and one lentil genotype, and two tomato cultivars, ‘Rutgers’ (susceptible) and ‘Nemadoro’ (resistant). Each plant (replication) was inoculated with 5000 eggs and second-stage juveniles (J2) of M. enterolobii and evaluated 65 days after inoculation, considering the following variables: gall index, egg mass index, number of eggs per g of roots, and reproduction factor. The experiments were conducted at two independent time points (summer and autumn/winter). Results showed that all 23 plant genotypes were susceptible to the nematode, with pea genotype ‘Itapuã’ being intolerant to infection. Cultivation of pulse crops has been steadily increasing both in Brazil and worldwide. Our research findings make a valuable contribution to the ongoing efforts to identify genetic resistance to nematode pathogens that can significantly affect the productivity of these crops. By identifying and developing resistant genotypes, pulse crop yields can be safeguarded, and sustainable agricultural practices can be supported.

Long-term benefit contribution of chemical and biological nematicide in coffee nematode management in soil microbial diversity and crop yield perspectives

The plant-parasitic root-knot nematode Meloidogyne exigua causes significant damage and is an important threat in Coffea arabica plantations. The utilization of plant-beneficial microbes as biological control agents against sedentary endoparasitic nematodes has been a longstanding strategy. However, their application in field conditions to control root-knot nematodes and their interaction with the rhizospheric microbiota of coffee plants remain largely unexplored. This study aimed to investigate the effects of biological control agent-based bioproducts and a chemical nematicide, used in various combinations, on the control of root-knot nematodes and the profiling of the coffee plant rhizomicrobiome in a field trial. The commercially available biological products, including Trichoderma asperellum URM 5911 (Quality), Bacillus subtilis UFPEDA 764 (Rizos), Bacillus methylotrophicus UFPEDA 20 (Onix), and nematicide Cadusafos (Rugby), were applied to adult coffee plants. The population of second-stage juveniles (J2) and eggs, as well as plant yield, were evaluated over three consecutive years. However, no significant differences were observed between the control group and the groups treated with bioproducts and the nematicide. Furthermore, the diversity and community composition of bacteria, fungi, and eukaryotes in the rhizosphere soil of bioproduct-treated plants were evaluated. The dominant phyla identified in the 16 S, ITS2, and 18 S communities included Proteobacteria, Acidobacteria, Actinobacteria, Ascomycota, Mortierellomycota, and Cercozoa in both consecutive years. There were no significant differences detected in the Shannon diversity of 16 S, ITS2, and 18 S communities between the years of data. The application of a combination of T. asperellum, B. subtilis, and B. methylotrophicus, as well as the use of Cadusafos alone and in combination with T. asperellum, B. subtilis, and B. methylotrophicus, resulted in a significant reduction (26.08%, 39.13%, and 21.73%, respectively) in the relative abundance of Fusarium spp. Moreover, the relative abundance of Trichoderma spp. significantly increased by 500%, 200%, and 100% at the genus level, respectively, compared to the control treatment. By constructing a co-occurrence network, we discovered a complex network structure among the species in all the bioproduct-treated groups. However, our findings indicate that the introduction of exogenous beneficial microbes into field conditions was unable to modulate the existing microbiota significantly. These findings suggest that the applied bioproducts had no significant impact on the reshaping of the overall microbial diversity in the rhizosphere microbiome but rather recruited selected microrganisms and assured net return to the grower. The results underscore the intricate nature of the rhizosphere microbiome and suggest the necessity for alternate biocontrol strategies and a re-evaluation of agricultural practices to improve nematode control by aligning with the complex ecological interactions in the rhizosphere.

The Main Compounds of Bio-Fumigant Plants and Their Role in Controlling the Root-Knot Nematode Meloidogyne incognita (Kofoid and White) Chitwood

Meloidogyne spp. are important parasitic nematodes affecting a variety of plants worldwide. We investigated the nematicidal properties of specific compounds found in bio-fumigant plants, particularly linalool, nonanal, methylene chloride, and 2-Decanal. Laboratory findings revealed that methylene chloride and 2-Decenal effectively reduced populations of second-stage juveniles (J2s). Additionally, the research explored the effects of tomato (Solanum lycopersicum L.) on M. incognita J2s, observing that tomato leaves significantly increased J2 mortality for all time measurements and different temperatures, while the opposite results were observed for root-stems. In the study, leaf treatment resulted in a maximum mortality response (MRmax) and half-maximal effective concentration (EC50) of approximately 100% and 4.0 µg/mg, respectively, at a temperature of 35 °C by week 8. In contrast, the root-stems treatment showed an MRmax of 13.5% and an EC50 of 3.0 ± 1.7 µg/mg. GC-MS analysis identified key compounds in tomato leaves and root-stems, such as α-pinene, d-limonene, and linalool. The results suggest that tomato leaves have potential as effective bio-fumigants for controlling root-knot nematodes.