First report of root-knot nematode, Meloidogyne javaniva, infecting Stachys byzantina on 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 & 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.

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 incognita, Infecting Buckwheat, Fagopyrum esculentum, in State of São Paulo, Brazil.

Buckwheat (Fagopyrum esculentum Moench) belongs to the Polygonaceae family and has been widely cultivated due to its high nutritional, nutraceutical, and medicinal properties. Brazil ranks seventh-largest producer, with 66,000 tons produced in 2018. Buckwheat is also valued for its adaptability as a cover crop, in grain fields of soybean (Glycine max (L.) Merr., maize (Zea mays L.), and sorghum (Sorghum bicolor (L.) Moench) (Görgen et al. 2016, Babu et al. 2018) especially in fields highly infested with plant-parasitic nematodes (PPN). PPN cause severe root damage, suppressing plant development and yield production. In October 2018, six samples of roots and soil were collected in symptomatic patches of buckwheat, in Guaíra SP (20° 19' 32"S 48° 13' 15.4"W). Samples were analyzed in the Nematology Laboratory (LabNema), UNESP, Jaboticabal, SP, BR. Plants presented symptoms of yellow leaves and galled and volume-reduced roots. Meloidogyne sp. was found, comprising 6,320 eggs and second-stage juveniles (J2s) from 10 g of root and 1,628 J2s in 100 cm³ of soil. Adult morphological characteristics, isoenzyme phenotype of esterase, and molecular analysis were performed to identify the Meloidogyne species. The perineal patterns presented high and trapezoidal dorsal arch (n=15), and the males showed a trapezoidal labial region, including a high head cap formed by a large round labial disc that is raised above the medial lips and centrally concave (n=15) (Eisenback and Hirscmann 1981). These characteristics are typical in Meloidogyne incognita (Kofoid and White, 1912) Chitwood, 1949 (Nascimento et al., 2020; Eisenback and Hirschmann 1981; Netscher and Taylor 1974). The enzymatic phenotype was performed with females (n=8), and the phenotype I1 was verified, described by Esbenshade and Triantaphyllou (1985) as typical for M. incognita. To confirm the species DNA samples were extracted from individual females (n=6) and PCR with specific primers for M. incognita (Mi-F 5'- GTGAGGATTCAGCTCCCCAG-3' and Mi-R 5'-ACGAGGAA CATACTTCTCCGTCC-3') and M. javanica (Treub) Chitwood 1949 (Fjav 5'-GGTGCGCGATTGAACTGAGC-3' and Rjav 5'-CAG GCCCTTCAGTGGAACTATAC-3') that amplify SCAR markers described by Meng et al. (2004) and Zijlstra et al. (2000), respectively, and specific primers for M. enterolobii Yang & Eisenback 1983 that amplify rDNA-IGS2 region (Me-F 5'-AACTTTTG TGAAAGTGCCGCTG-3' and Me-R 5'-TCAGTTCAGGCAGG ATCAACC-3') described by Long et al. (2006) were tested. A fragment of 955 pb DNA size was amplified in Mi-F/R primer, which confirmed the M. incognita identification (Meng et. al., 2004). The original population was used to execute pathogenicity test. In a greenhouse, single buckwheat seeds (cv. IPR 91 Baili) were sown in six 5L pots filled with autoclaved-soil and inoculated with 3,000 eggs and J2s per pot (n=6) and control (n=6). After 60 days, the nematodes were extracted from roots and the M. incognita was confirmed. An average of 15,738 eggs and J2s were recovered, (reproductive factor = 5.24), which confirmed buckwheat as a host to M. incognita. The inoculated plants showed symptoms as those observed in the field. No symptom or nematode was noted on the control. Meloidogyne incognita has been reported causing high damage to the F. esculentum in California (Gardner and Caswell-Chen 1994) plus several crops in Brazil (Nascimento et al., 2020). However, this is the first report of this nematode infecting buckwheat in Brazil. Given the importance of buckwheat in Brazil, with extensive use as forage, cover crop, and its nutritional properties, this report is essential to specific management measures are adopted to avoid further losses.

First Report of Root-Knot Nematode Meloidogyne enterolobii Infecting Sweetpotato in the State of Rio Grande do Norte, Brazil.

The sweetpotato (Ipomoea batatas L., Convolvulaceae family) originated in Latin America and is currently cultivated worldwide. The storage roots, rich in calories, have made this crop one of the main caloric sources for low-income populations, especially in developing countries. Brazil annually produces about 805,000 tons, with the Northeast region responsible for 34% of this production (Albuquerque et al. 2020). In October 2019, sweetpotato plants cv. Campina, from a field in the region of Touros, state of Rio Grande do Norte (RN), Brazil (5°12'31"S 35°34'42"W), presented deformed storage roots, with galls, typical of root-knot nematodes. The roots were sent to the Nematology Laboratory (LabNema) where 14,032 eggs and 3,312 second-stage juveniles (J2s) of Meloidogyne sp., in 10 g of roots, were recovered. The species of adults was identified through morphological, biochemical, and phylogenetic analysis. The perineal region of females (n = 10) presented an oval shape, with a high and semi-trapezoidal dorsal arch and streak-free perivulval region. The labial region of males (n=10) presented high and rounded head cap, labial region slightly set off from the body, without annulations. The morphological characters were compatible with the original description of Meloidogyne enterolobii (Yang and Eisenback 1983). The phenotype of esterase isoenzymes showed two major bands (VS1-S1) also characteristic of M. enterolobii (Esbenshade and Triantaphyllou 1985). Sequences of 18S rDNA (~1200bp) of individual females (Holterman et al. 2006) obtained from sweetpotatoes before (SPme1 and 2) and after inoculation (SPme3 and 6), and from guava, used as M. enterolobii species control, were submitted to Bayesian analysis. The sequences presented genetic diversity among them resulting from seven SNPs (Single Nucleotide Polymorphism) and 99.4 to 99.9% identity with M. enterolobii sequences deposited in the NCBI GenBank (accession numbers MW209034-MW209039). The pathogenicity test was carried out under greenhouse conditions, in which 3,000 eggs and J2s from the original population isolated of M. enterolobii were inoculated in sweetpotato seedlings cv. Campina (n = 6). After three months, the roots presented galls and deformations typical of root-knot nematodes, while non-inoculated plants did not present any symptoms. An average of 15,900 eggs and J2s of M. enterolobii (RF = 5.3) were recovered from the roots, proving that sweetpotatoes were a host of this species. Meloidogyne enterolobii is known to cause great damage to sweetpotato (Ye et al. 2020). In Brazil, Meloidogyne nematode had been reported once, isolated from a sweetpotato field in the Ceara state and the species suggested by the authors according to esterase electrophoresis was M. enterolobii. Nonetheless, the authors did not present taxonomic, isoenzyme phenotypes and molecular species identification integratively, nor included pathogenicity tests (Silva et al. 2016). Therefore, it is the first time that M. enterolobii, with reliable identification by different methods, including sequencing, was detected in commercial sweetpotato fields in the RN state and in Brazil. The local farmers reported that this nematode deforms the storage roots which make them useless for commercialization, resulting in minimal losses of 50% of production in the infested areas. Furthermore, as sweetpotatoes are vegetatively propagated, the spread of this nematode through planting material is favored. Considering the importance of this crop in Brazil, this report is essential for control measures of this pathogen to be taken in order to avoid its spread to other regions.

First Report of Meloidogyne incognita Infecting Stachys byzantina in Brazil.

Stachys byzantina C. Koch (Lamiaceae alt. Labiatae), commonly known as lamb's ear, is an important medicinal plant with anti-inflammatory, antitumor, anticancer, antispasmodic, sedative and diuretic properties (Asnaashari et al. 2010). This plant is widely consumed in Europe and Asia as aromatic teas. In Brazil, it is an unconventional food plant, nonetheless, its medicinal properties have been recognized as well as its production. In May 2019, in a Sao Paulo State municipality, Jaboticabal, (21°14'38.7"S 48°17'10.6"W), S. byzantina plants presented reduced growth and chlorotic leaves associated with root galls. In the phytopathological clinic, 7,983 eggs and juveniles of Meloidogyne sp. were counted in 10 g of the plant roots. In 100 cm³ of soil surrounding the plant, 532 second-stage Meloidogyne sp. juveniles (J2) were found. Morphological, enzymatic and molecular identification of the nematode species found were performed (Fig. S1). For morphological analysis, perineal pattern of females (n = 10) and labial region of males (n = 10) were analyzed. In the perineal region of females, a high and trapezoidal dorsal arch with thick striations was observed, whereas the males presented the trapezoidal labial region with the prominent labial disc in relation to the sub-median lips and transverse streaks in the head region, typical characteristics of M. incognita (Kofoid and White, 1919) Chitwood 1949. (Netscher and Taylor 1974; Eisenback and Hirschmann 1981). The esterase enzyme profile, obtained individually from 8 females, was compatible with phenotype I1 [Rm (x100) = 46.25], also associated with M. incognita (Esbenshade and Triantaphyllou 1985). Molecular analysis was realized (n = 3) by applying the primers Finc/Rinc (Zijlstra et al. 2000) in the DNA of individual females, which resulted in the amplification of an amplicon of 1200 bp specific for M. incognita. Pathogenicity testing was conducted in a greenhouse by inoculation of 5,000 eggs and juveniles from the original population into S. byzantina seedlings (n = 4). After 90 days, the inoculated plants, unlike the non-inoculated ones, exhibited symptoms similar to those initially observed in the field. The nematodes were extracted from the roots of the inoculated plants, quantified, and the identity of M. incognita was confirmed. The average reproductive factor obtained was 136.6, confirming the pathogenicity of M. incognita to S. byzantina. Thus, this is the first report of M. incognita associated with S. byzantina in Brazil and in the world. Lamb's ear is a horticultural plant, and its high reproductive factor to M. incognita can also result in damage to the subsequent crops. In addition, Lamb's ear is propagated vegetatively and this favors the spread of nematodes to other areas. This new report is important in order to alert producers to realize the proper management of this nematode in S. byzantina.