Development and Genetic Characterization of Peanut Advanced Backcross Lines That Incorporate Root-Knot Nematode Resistance From Arachis stenosperma

Crop wild species are increasingly important for crop improvement. Peanut (Arachis hypogaea L.) wild relatives comprise a diverse genetic pool that is being used to broaden its narrow genetic base. Peanut is an allotetraploid species extremely susceptible to peanut root-knot nematode (PRKN) Meloidogyne arenaria. Current resistant cultivars rely on a single introgression for PRKN resistance incorporated from the wild relative Arachis cardenasii, which could be overcome as a result of the emergence of virulent nematode populations. Therefore, new sources of resistance may be needed. Near-immunity has been found in the peanut wild relative Arachis stenosperma. The two loci controlling the resistance, present on chromosomes A02 and A09, have been validated in tetraploid lines and have been shown to reduce nematode reproduction by up to 98%. To incorporate these new resistance QTL into cultivated peanut, we used a marker-assisted backcrossing approach, using PRKN A. stenosperma-derived resistant lines as donor parents. Four cycles of backcrossing were completed, and SNP assays linked to the QTL were used for foreground selection. In each backcross generation seed weight, length, and width were measured, and based on a statistical analysis we observed that only one generation of backcrossing was required to recover the elite peanut’s seed size. A populating of 271 BC3F1 lines was genome-wide genotyped to characterize the introgressions across the genome. Phenotypic information for leaf spot incidence and domestication traits (seed size, fertility, plant architecture, and flower color) were recorded. Correlations between the wild introgressions in different chromosomes and the phenotypic data allowed us to identify candidate regions controlling these domestication traits. Finally, PRKN resistance was validated in BC3F3 lines. We observed that the QTL in A02 and/or large introgression in A09 are needed for resistance. This present work represents an important step toward the development of new high-yielding and nematode-resistant peanut cultivars.

Meta-analysis of the field efficacy of seed- and soil-applied nematicides on Meloidogyne incognita and Rotylenchulus reniformis across the U. S. Cotton Belt

Meta-analysis was used to compare yield protection and nematode suppression provided by two, seed- and two, soil-applied nematicides against Meloidogyne incognita and Rotylenchulus reniformis on cotton across three years and several trial locations in the United States Cotton Belt. Nematicides consisted of thiodicarb- and fluopyram-treated seed, aldicarb and fluopyram applied in-furrow and combinations of the seed treatments and soil-applied fluopyram. The nematicides had no effect on nematode reproduction or root infection but had a significant impact on seed cotton yield response (¯D) with an average increase of 176 and 197 kg/ha relative to the nontreated control in M. incognita and R. reniformis infested fields, respectively. However, because of significant variation in yield protection and nematode suppression by nematicides, five or six moderator variables [cultivar resistance (M. incognita only), nematode infestation level, nematicide treatment, application method, trial location, and growing season] were used depending on nematode species. In M. incognita infested fields, greater yield protection was observed with nematicides applied in-furrow and seed-applied + in-furrow than solo seed-applied nematicide applications. Most notably of these in-furrow nematicides were aldicarb and fluopyram (>131 g/ha) with or without a seed-applied nematicide compared to thiodicarb. In R. reniformis infested fields, moderator variables provided no further explanation of the variation in yield response by nematicides. Furthermore, moderator variables provided little explanation of the variation in nematode suppression by nematicides in M. incognita and R. reniformis infested fields. The limited explanation by the moderator variables on the field efficacy of nematicides in M. incognita and R. reniformis infested fields demonstrates the difficulty of managing these pathogens with nonfumigant nematicides across the U. S. Cotton Belt.

First Report of Meloidogyne javanica on Globe Artichoke in Florida, USA

Globe artichoke (Cynara cardunculus var. scolymus L.) is native to the Mediterranean region and cultivated worldwide for its edible flower buds and the medicinal value of its leaves (Pignone and Sonnante 2004). In 2019, artichokes were planted on 29 km2 predominantly in California, with a yield of over 100 million kg (USDA 2020). It has been grown as a specialty crop in Florida since 2017 (Agehara 2017a). Meloidogyne spp. (root-knot nematodes/RKNs) can lead to yield losses to artichoke (Greco et al. 2005). In June 2020, artichokes (cv. Imperial Star) with stunting, wilting, and galled-root symptoms were observed in a research field with sandy soil located at the University of Florida Gulf Coast Research and Education Center (UF/GCREC), Wimauma, Florida. The goal of this report was to identify the RKN species collected from two symptomatic artichoke roots. Morphological measurements (mean, standard deviation and range) of 15 second-stage juveniles (J2s) included body length = 409.1 ± 31.6 (360.3 - 471.3) µm, body width = 15.4 ± 1.6 (12.4 - 18.8) µm, and stylet length = 14.7 ± 0.7 (13.9 -16.1) µm. Perineal patterns of five matured females had a high dorsal arch and double lateral lines. Morphological characteristics of the RKN cultures were consistent with the description of M. javanica (Eisenback and Triantaphyllou 1991). DNA was extracted respectively from two RKN females isolated from the diseased artichoke roots. The nematode species was confirmed with primers Fjav/Rjav and resulted in ≈ 670 bp fragment (Zijlstra et al. 2000). The COXII region of mtDNA was amplified by C2F3/1108 (Powers and Harris 1993), and the sequencing results were submitted to the NCBI with GeneBank Accession No. MZ397905. The molecular sequences had 100% identity with M. javanica in COXII (MK033440 and MK033439). The pathogenicity test was conducted in the greenhouse at UF/GCREC from May to August 2021 (temperature = 26.7 ± 4.1°C, relative humidity = 83.9 ± 14.6 %). Each of the ten 6.5-in-diameter plastic pots containing 3.8-L pasteurized soil was seeded with one artichoke seed. Five pots were inoculated with 5000 eggs of the field RKN cultures 4-week after planting, and five pots served as the untreated control. Two months after inoculation, galled symptoms were only observed in inoculated plants with an average gall index (Bridge and Page 1980) of 6.2 ± 2.2; 99,240 ± 72,250 eggs were extracted from each root system, and the nematode reproduction factor was 19.9 ± 14.4. Meloidogyne spp. has been reported on artichoke in Europe, Asia, and South America (Greco et al. 2005). This is the first report of RKN on artichoke in the United States. Meloidogyne javanica caused severe root gall symptoms and visible aboveground damage in the form of chlorosis, stunting, and wilting of artichoke planted at the UF/GCREC research farm. Meloidogyne javanica is the predominant RKN species at the UF/GCREC research farm and one of the most common RKNs in Florida (Gu and Desaeger 2021). Artichoke is a new crop in Florida, and RKNs is likely to be one of the main soilborne problems for its production in the state. Its long growing season (October - May) (Agehara 2017b) allows for high nematode reproduction rates. Several new growers have already reported RKN as a problem in their fields. For artichoke to become a commodity in Florida, managing RKNs will be critical. This report provides new information on the risk that RKNs pose to artichoke, a newly established specialty crop in Florida.

First Report of Meloidogyne graminicola on Tomato (Solanum lycopersicum) in Hainan of China

Tomato (Solanum lycopersicum) is an important vegetable crop in Hainan province, Southern China. In this area, rice and tomato rotation is the most common way for tomato cultivation. During March of 2021, in a field of Yazhou District, Sanya City, Hainan Province, leaves of some tomato plants (cv. Jinsheng) turned yellow, although there were no obvious dwarf plants observed. The tomato plants with yellow leaves exhibiting obvious galls on the roots were collected. Several females and gelatinous egg masses of Meloidogyne spp. were found inside the cortex of the root galls after dissection. The perineal patterns of females (n=12) were dorsal-ventrally oval with low and round dorsal arches, lacking obvious lateral lines. Most of the striae were smooth and sometimes short and irregular striae were observed within them. Morphological measurements of females (n=20) included body length (L) = 569.2 ± 53.6 (457.6 - 662.7) µm, body width (BW) = 342.7 ± 69.8 (245.5 - 457.9) µm, stylet = 11.8 ± 0.7 (10.5 - 13.3) µm, dorsal pharyngeal gland orifice to stylet base (DGO) = 4.0 ± 0.2 (3.7 - 4.6) µm, vulval slit length = 24.1 ± 3.7 (16.7 - 30.7) µm, and vulval slit to anus distance = 16.0 ±1.9 (12.6 - 19.3) µm. The second-stage juveniles (J2s, n=20) had the following morphological characters: L = 440.6 ± 26.7 (395.7 - 488.3) µm, BW = 15.9 ± 1.0 (14.5 - 17.9) µm. stylet = 13.5 ± 0.8 (12.3 - 14.9) µm, tail length = 69.5 ± 3.7 (65.4 - 76.9) µm, hyaline tail terminus = 21.0 ± 2.1 (17.3 - 24.9) µm. These morphological characters matched the original description of Meloidogyne graminicola (Golden and Birchfield, 1968). Ten individual females were transferred to ten different tubes for DNA extraction. The species-specific primers Mg-F3 (5'-TTATCGCATCATTTTATTTG-3') and Mg-R2 (5'-CGCTTTGTTAGAAAATGACCCT-3') were used for the identification of M. graminicola (Htay et al. 2016). For the ten DNA samples, a 369 bp fragment was amplified by this pair of primers, confirming their identities as M. graminicola. The mitochondrial DNA (mtDNA) region between COII and the lRNA gene was amplified using primers C2F3 (5’-GGTCAATGTTCAGAAATTTGTGG-3’) and 1108 (5’-TACCTTTGACCAATCACGCT-3’) (Powers and Harris, 1993). A DNA fragment of 531 bp was obtained and the sequence (GenBank Accession No. MZ576221) was 99.8% identical to the sequences of M. graminicola (GenBank Accession Nos. MH033621, MK616527, and MG356945). Part of the rDNA spanning ITS1, 5.8S gene, and ITS2 was amplified with primers 18S (5’-TTGATTACGTCCCTGCCCTTT-3’) and 26S (5’-TTTCACTCGCCGTTACTAAGG-3’) (Vrain et al. 1992). The sequences from the ITS region were 790 bp (GenBank Accession No. MZ312595) and were all 100% identical to the known sequences of M. graminicola (GenBank Accession Nos. MF320126, HM623442, and KY020414). In glasshouse tests, six 30-day-old tomato plants (cv. Jinsheng) were individually transplanted in pots (V sand :V soil = 3:1) and inoculated with 1500 J2s hatched from the egg masses of collected M. graminicola samples per plant. Two non-inoculated tomato plants served as negative controls. After 50 days, inoculated plants had galled roots similar to those encountered in the field and there were J2s and eggs within the galls. The nematode reproduction factor (RF = final population/initial population) was 5.3. No symptoms were observed on control plants. These results confirmed the nematode’s pathogenicity on tomato. To our knowledge, this is the first time of a natural infection of tomato with M. graminicola in China.

First report of Meloidogyne enterolobii infecting cassava (Manihot esculenta) resulting in root galling damage in Africa

Meloidogyne enterolobii is a highly polyphagous tropical species of root knot nematode. It has been recorded to be causing major damage to a range of economically important crops and is increasingly recorded from new locations. The morphological similarity and overlap of characteristics with other commonly occurring species, especially M. incognita, has confused its diagnosis using morphometrics. Cassava (Manihot esculenta) is an important crop across the tropics, including Africa, where it is among the most important root and tuber crop for food security. Cassava can be heavily infected by root knot nematodes, which can incur heavy production losses. The main species known to affect cassava are M. incognita and M. javanica (Coyne and Affokpon, 2018). With the exception of one report of M. enterolobii morphologically identified from cassava roots during a survey in Brazil (Rosa et al., 2014), there is no record with molecular confirmation of it infecting the crop. In the absence of any molecular or isozyme confirmation, diagnosis of M. enterolobii is difficult to determine. In the current study, the species responsible for substantial galling damage (Fig. 1A) on several cassava roots growing in Ibadan, Nigeria (7°22′39″ N; 3°54′21″ E) were diagnosed. DNA isolated from juveniles recovered using a modified Baermann method (Hooper, 1986) from these roots was used for PCR amplification of the mitochondrial Nad5 using primer pair, NAD5F2 (5’-TATTTTTTGTTTGAGATATATTAG-3’) and NAD5R1 (5’-CGTGAATCTTGATTTTCCATTTTT-3’). The 515 bp PCR DNA product was sequenced on both strands (GenBank Accession No. MW965454) and found to be 100% identical to M. enterolobii with those in the DNA sequence database (KU372358, KU372359) (Janssen et al., 2016; Kolombia et al., 2017). In addition, M. incognita was also recovered from the galled roots and identified using the same primers (GenBank Accession No. MW965455) indicating a combined species infection (Fig. 2). Cultures of M. enterolobii, developed from single egg masses were maintained on tomato plants and used to assess infection on cassava in 10 L pots filled with steam sterilized loam soil in the screenhouse. Cassava cv. IITA-TMS-IBA070593 cuttings planted in June, 2018 and repeated in April, 2019 were inoculated with 1,000 juveniles per pot at three weeks after planting, and then maintained for four months before quantifying the nematode densities in both roots and soil. Nematode reproduction factor (RF), calculated from total nematode densities (n=8) from soil and roots was as high as 44.3, compared to uninoculated controls. Molecular diagnostics of M. enterolobii, as above, confirms unequivocally the host status of cassava to this nematode. This study reports for the first time the infection of cassava by M. enterolobii under field conditions in Africa and for the first time demonstrates the host suitability of cassava to this nematode (Fig. 1B). M. enterolobii is among the most commonly occurring root-knot nematode species in West Africa (dos Santos et al., 2019). It is therefore anticipated that M. enterolobii has long been infecting, especially in West Africa, but has been overlooked due to its morphological similarity with M. incognita. Given the high reproductive ability of M. enterolobii on cassava and its highly aggressive nature on a range of crops, it is likely that it is causing, or will result in, high levels of losses on cassava in Africa.

Haemonchus contortus Transthyretin-Like Protein TTR-31 Plays Roles in Post-Embryonic Larval Development and Potentially Apoptosis of Germ Cells

Transthyretin (TTR)-like proteins play multi-function roles in nematode and are important component of excretory/secretory product in Haemonchus contortus. In this study, we functionally characterised a secretory transthyretin-like protein in the barber’s pole worm H. contortus. A full-length of transthyretin-like protein-coding gene (Hc-ttr-31) was identified in this parasitic nematode, representing a counterpart of Ce-ttr-31 in Caenorhabditis elegans. High transcriptional levels of Hc-ttr-31 were detected in the egg and early larval stages of H. contortus, with the lowest level measured in the adult stage, indicating a decreased transcriptional pattern of this gene during nematode development. Localisation analysis indicated a secretion of TTR-31 from the intestine to the gonad, suggesting additional roles of Hc-ttr-31 in nematode reproduction. Expression of Hc-ttr-31 and Ce-ttr-31 in C. elegans did not show marked influence on the nematode development and reproduction, whereas Hc-ttr-31 RNA interference-mediated gene knockdown of Ce-ttr-31 shortened the lifespan, decreased the brood size, slowed the pumping rate and inhibited the growth of treated worms. Particularly, gene knockdown of Hc-ttr-31 in C. elegans was linked to activated apoptosis signalling pathway, increased general reactive oxygen species (ROS) level, apoptotic germ cells and facultative vivipary phenotype, as well as suppressed germ cell removal signalling pathways. Taken together, Hc-ttr-31 appears to play roles in regulating post-embryonic larval development, and potentially in protecting gonad from oxidative stress and mediating engulfment of apoptotic germ cells. A better knowledge of these aspects should contribute to a better understanding of the developmental biology of H. contortus and a discovery of potential targets against this and related parasitic worms.

Hexapeptides from mammalian inhibitory hormone hunt activate and inactivate nematode reproduction

Increased reproduction (x3) of the entomopathogenic nematode Steinernema siamkayai occurred when exposed to one synthetic peptide (IEPVFT), while the fecundity of worms exposed to hexamer (KLKMNG) was reduced (x0.5). These hexamers were opposite ends of a 14 amino acid (aa) synthetic peptide KLKMNGKNIEPVFT (EPL030). The bioactivity of the hexamers is surprising it is a scrambled-sequence control of another peptide, MKPLTGKVKEFNNI (EPL001) which are bioinformatically obscure. EPL001 emerged from a physicochemical fractionation aimed at finding a postulated hormone that is reproductively related and tissue-mass reducing and has antiproliferative effects on human prostate cancer cells and rat bone marrow cells in vitro. Intracerebroventricular infusion of EPL001 in sheep was associated with elevated growth hormone in peripheral blood and reduced prolactin. The highly dissimilar EPL001 and EPL030 nonetheless have the foregoing biological effects in common in mammalian systems, while being divergently pro- and anti-fecundity respectively in the nematode Caenorhabditis elegans. Immunoprecipitation of EPL001 using an anti-EPL001 antibody suggests it encodes the sheep neuroendocrine prohormone secretogranin II (sSgII). Using bespoke bioinformatics with six sSgII residues deduced bioactivity to key aa: MKPLTGKVKEFNNI. Peptides more potent as cell inhibitors than EPL001 suggest a stereospecific bimodular tri-residue signature (i.e. simultaneous accessibility for binding of two specific trios of aa side chains, MKP & VFN). An evolutionarily conserved receptor is conceptualised as having dimeric binding sites, each with ligand-matching bimodular stereocentres. Sequence analysis and computational modelling suggest the activity of the control peptide EPL030 and its N- and C-terminal hexapeptide progeny is due the novel hormonal motif MKPVFN.

Relative Efficacy of Some Products Against Meloidogyne Javanica (Treub) Chitwood on Tomato Under Greenhouse Conditions

A pot experiment was conducted to compare the efficacy of some products i.e., Stanes Bio Nematon®, Soft Guard®, Paecilomyces lilacinus, Trichoderma longibranchiatum, camel and goat manures against Meloidogyne javanica on tomato under greenhouse conditions. Based on nematode reproduction, indices of galls and egg masses, the six materials were grouped into four classes from the relatively highest efficacy of control (goat manure) to the relatively low efficacy of control (P. lilacinus). Bangladesh J. Bot. 50(3): 709-712, 2021 (September)

Meloidogyne enterolobii development and reproduction in tomato plants treated with resistance inducers

Summary Meloidogyne enterolobii is a species capable of overcoming plant resistance moderated by the Mi-1 gene, which is effective against most species of root-knot nematode. This study evaluated the effect of induced resistance in tomato plants (Solanum lycopersicum ‘H-9553’) with the Mi-1 gene against the development and reproduction of M. enterolobii. Seedlings of tomato ‘H-9553’ were transplanted into pots, inoculated with 2000 eggs and second-stage juveniles (J2) of M. enterolobii and treated with Acibenzolar-S-Methyl, Bacillus subtilis, B. subtilis + B. licheniformis + Trichoderma longibrachiatum and extract of Reynoutria sachalinensis. The plants were collected at 5, 10, 15, 20, 25 and 30 days after inoculation (DAI) for the analyses of nematode penetration and development, and at 30 DAI for nematode reproduction. The use of B. subtilis increased fresh root weight when compared to the other treatments (20 DAI). There was a reduction in penetration of J2 in the roots of plants subjected to different resistance inducers. The population density of M. enterolobii was significantly reduced only when plants were treated with R. sachalinensis, indicating it as a potential resistance-inducing agent in tomato plants.

First report of southern root-knot nematode, Meloidogyne incognita, infecting Isatis indigotica Fortune in Anhui Province, China

Isatis indigotica Fortune, widely cultivated in China, is an important Chinese herbal medicine, mainly used to treat cold and fever. In October 2020, galls (Fig. 1), as many as 65 per root, were observed on the roots of I. indigotica in Taihe, Anhui Province, China (117°21'19.5"N, 32°57'59.5"E), and samples were taken. The infected plants were weak, and the leaves are wilting. Second-stage juveniles (J2s) were dissected from the egg masses released by females. Excretory pores of females were located nearby median bulb (Fig. 2A). The dorsal arch of the perineal pattern (n = 10) of the female was elliptical, and the dorsal arch was relatively high with smooth to wavy lines (Fig. 2B). Morphometrics of females (n=10): body length (L) = 595.5 ± 24.0 (570.0-620.5) μm, body width (W)= 350.5 ± 30.0 (320.0-390.5) μm, stylet length = 13.6 ± 0.7 (12.1-15.4) μm (Fig. 2A), and the distance from dorsal esophageal gland orifice to base of stylet (DGO) = 3.5 ± 0.2 (2.8-4.0) μm (Fig. 2B). J2s (n = 20) had the following characteristics: L = 383.2 ± 12.5 (337-430) μm (Fig. 2C), a = 22.0 ± 1.1 (20.3-24.4) μm, c = 8.4 ± 0.5 (7.5-10.5) μm, stylet length = 12.4 ± 1.5 (10.1-14.6) μm, DGO = 2.9 ± 0.6 (2.0-3.6) μm (Fig. 2D), tail length = 39.5 ±3.4 (32.0-48.5) μm and hyaline tail terminus = 10.5 ± 0.5 (9.5-11.2) μm (Fig.1E). There were four lines on the lateral field of J2s (Fig. 2F). Females and J2s obtained from galls had uniform morphological and molecular characteristics were confirmed to be Meloidogyne incognita. Live J2s were detected in all soil samples with a mean of 120 ± 15 J2s/100 ml of soil. Five 4-week-old I. indigotica plantlets, grown in pots (500cm3) with sterilized soil were inoculated with 1000 J2s from egg masses per pot and5 non-inoculated pots were used as control. Plants were well maintained under 25 ± 3°C in the greenhouse. Three plants were gently removed from the pots 30 days after inoculation, and an average of 50 galls per root was observed on the roots, and the resulting nematode reproduction factors (RF = final egg density ÷ 1,000, initial egg density) of 3.2, suggested that I. indigotica is a good host for M. incognita (Mojtahedi, 1988). There were no significant differences in main measurements and morphological characteristics between the Taihe population of M. incognita and that represented in "CIH descriptions of plant-parasitic nematodes" (Orton Williams, 1973). DNA was extracted from 5 single J2s, and ITS and 18S rDNA gene was amplified using the primer pair 18S/26S and 18s1.2a/18sr2b (Bernard et al. 2010; Vrain et al. 1992). The sequence of 18S rDNA (MW875892) was submitted to GenBank. Comparisons showed a sequence identity of greater than 99.8% for Meloidogyne incognita (MF177719.1). The rDNA sequences of M. incognita, M. hapla, M. javanica and M. arenaria are so homologous that rDNA-based differentiation is difficult. The SCAR primers can successfully distinguish M. incognita, M. hapla, M. javanica and M. arenaria. Five species-specific primer sets (Finc/Rinc; MORF-F/MTHIS-R; Jmv-F/Jmv-R; Far/Rar and Fjav/Rjav, Stanton et al. 1997; Wishart et al. 2002; Zijlstra et al. 2000) were used to species-specifically distinguish within the genus. The results (＋, ＋, －, －, －) proved that the Taihe population belonging to M. incognita. To our knowledge, this is the first report of M. incognita parasitizing I. indigotica. This finding may be important to medicinal plant industry, since M. incognita is one of the most harmful nematode pests in the world and would cause severe damage to I. indigotica.