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 Meloidogyne javanica Infecting Strawberry (Fragaria × ananassa) in the United States.

Strawberry (Fragaria × ananassa) is native to temperate regions. However, it has been produced in tropical areas, as a seasonal crop including in Florida, USA during the winter months. In March 2022, root galls resembling those induced by root-knot nematodes (Meloidogyne spp.) were observed in declining strawberry plants 'WinterstarTM FL 05-107' growing in an organic-certified research site in Hillsborough County, Florida, USA. To our knowledge, M. hapla is the only root-knot species reported to infect strawberry in Florida. Preliminary molecular analyses, including newly synthesized DNA sequences (TW81/AB28 = OQ469833 - OQ469836; D2A/D3B= OQ473043 - OQ473047) using extracted nematode females from the strawberry roots, initially identified the RKN as M. javanica. Nematode species confirmation was further performed using the morphology of the female perineal patterns and isozyme analysis, mainly esterase (EST) and malate dehydrogenase (MDH), DNA sequencing, (NAD5-F/NAD5-R) and the SCAR primer set (Fjav/Rjav), species-specific for M. javanica. Isozyme analyses, EST= J3, which is specific for M. javanica and MDH=N1, as well as the morphology of female perineal patterns, agreed with data previously reported for M. javanica. A pathogenicity test on strawberry 'WinterstarTM FL 05-107' transplants was performed using 10,000 eggs of the original M. javanica population, which induced galls on strawberry plants (Gall index, GI = 4.1) with egg masses clearly visible outside of the roots, producing an average of 1,344 eggs/gram of fresh root and 9,201 ± 4,206 eggs/root system. No galls or egg masses were observed on non-inoculated plants. Tomato 'HM 1823' was used as a control for the viability of the inoculum and showed numerous galls and egg masses (GI=5.0;). The newly obtained DNA sequences using NAD5-F/NAD5-R (OQ474970 - OQ474972) were compared with other sequences available in the GenBank and were shown to be 100% identical to five M. javanica populations from Polk County, Florida, USA (OM418745 - OM418749) and the complete mitochondrion genome of M. javanica (NC026556). To our knowledge, this is the first report of M. javanica infecting strawberry in the United States.

Feeding Selectivity of Aphelenchoides besseyi and A. pseudogoodeyi on Fungi Associated with Florida Strawberry.

Aphelenchoides besseyi and A. pseudogoodeyi are foliar nematodes associated with commercial strawberry production in Florida, United States. The reproductive and feeding habits of these two nematode species were assessed on Florida isolates of the fungi Botrytis cinerea, Colletotrichum gloeosporioides, Macrophomina phaseolina, and Neopestalotiopsis rosae, which are pathogenic to strawberry, and nonpathogenic isolates of Fusarium oxysporum and Monilinia fructicola grown on potato dextrose agar in Petri dishes. Each culture was inoculated with six specimens of mixed life stages of A. besseyi or A. pseudogoodeyi and incubated at 24°C under axenic and nonaxenic conditions 23 and 31 days after inoculation, respectively. A. besseyi reproduction rates were greater on strawberry-pathogenic isolates of B. cinerea, C. gloeosporioides, and N. rosae than on the nonpathogenic isolates of F. oxysporum and M. fructicola. In contrast, reproductive rates of A. pseudogoodeyi did not vary among fungi cultures. For both nematode species, M. phaseolina was a poor host because it did not produce mycelium on the media used. Our findings indicate that A. besseyi is more selective in its fungal-feeding preference than A. pseudogoodeyi. Additionally, A. pseudogoodeyi eggs and juveniles were significantly more numerous than adults. Yet, for A. besseyi, adult stages were more abundant. Fungi aid in the maintenance of soil-dwelling populations of these two nematode species. Removing fungus-infected strawberry plant residues is both a desirable and effective management practice to limit A. besseyi in central Florida commercial strawberry fields.

Temperature Effects on Development of Meloidogyne Enterolobii and M. Floridensis.

Meloidogyne enterolobii and M. floridensis are virulent species that can overcome root-knot nematode resistance in economically important crops. Our objectives were to determine the effects of temperature on the infectivity of second-stage juveniles (J2) of these two species and determine differences in duration and thermal-time requirements (degree-days [DD]) to complete their developmental cycle. Florida isolates of M. enterolobii and M. floridensis were compared to M. incognita race 3. Tomato cv. BHN 589 seedlings following inoculation were placed in growth chambers set at constant temperatures of 25°C, and 30°C, and alternating temperatures of 30°C to 25°C (day-night). Root infection by the three nematode species was higher at 30°C than at 25°C, and intermediate at 30°C to 25°C, with 33%, 15%, and 24% infection rates, respectively. There was no difference, however, in the percentages of J2 that infected roots among species at each temperature. Developmental time from infective J2 to reproductive stage for the three species was shorter at 30°C than at 25°C, and 30°C to 25°C. The shortest time and DD to egg production for the three species were 13 days after inoculation (DAI) and 285.7 DD, respectively. During the experimental timeframe of 29 d, a single generation was completed at 30°C for all three species, whereas only M. floridensis completed a generation at 30°C to 25°C. The number of days and accumulated DD for completing the life cycle (from J2 to J2) were 23 d and 506.9 DD for M. enterolobii, and 25 d and 552.3 DD for M. floridensis and M. incognita, respectively. Exposure to lower (25°C) and intermediate temperatures (30°C to 25°C) decreased root penetration and slowed the developmental cycle of M. enterolobii and M. floridensis compared with 30°C.

Susceptibility of Flordaguard peach rootstock to a resistant-breaking population of Meloidogyne floridensis and two populations of Meloidogyne arenaria.

Cultivar Flordaguard is suggested as a root-knot nematode (RKN) resistant rootstock for Florida peaches, however, RKN disease has been observed on this rootstock in peach orchards. Our goal was to confirm whether the RKN resistance breaking isolates of M. floridensis and M. arenaria indeed could infect and reproduce on the peach rootstock cv. Flordaguard in both laboratory and field studies. Root galling occurred on all peach cultivars evaluated including Flordaguard, Flordaglo, Okinawa, and Lovell, in the presence of the RKN resistance-breaking isolates of M. floridensis (MfGnv14) and two M. arenaria isolates (Ma1 and Ma2). These rootstocks showed varying degrees of susceptibility (to a lesser extent in Okinawa) to these three RKN resistance-breaking isolates. The importance of nematode inoculum concentrations in differentiating between resistance and susceptible plants was demonstrated, and thus are an important factor to consider in nematode resistance breeding programs. In host differential tests the peach-originated isolates of M. floridensis and M. arenaria behaved similarly with the vegetable-originated isolates of M. floridensis on tomato, peanut, watermelon, and tobacco, but showed variable host responses on cotton and pepper. The two M. arenaria isolates from peach reproduced on pepper but not on peanut. To our knowledge this is the first report of M. arenaria race 3 infecting Flordaguard and pepper in Florida. Soil and root samples collected from cv. Flordaguard infected trees at two commercial peach orchards showed that M. floridensis and M. arenaria were established on the rootstock.

Reproduction of Meloidogyne enterolobii on selected root-knot nematode resistant sweetpotato (Ipomoea batatas) cultivars.

The ability of Meloidogyne enterolobii to reproduce on selected sweetpotato (Ipomoea batatas) cultivars (Beauregard, Covington, Evangeline, Hernandez, and Orleans (LA 05-111)) was evaluated in two greenhouse experiments, each with 10 replicates. All cultivars, except Beauregard (control) and Orleans, were reported previously as moderately resistant or resistant to M. incognita, Fusarium oxysporum f. sp. batatas, and Streptomyces ipomoeae. Plants were inoculated with M. enterolobii (5,000 eggs/plant) and arranged in a completely randomized design in a greenhouse with an average daily temperature of 24.8°C. Galls and egg masses per root system (0-5 scale), eggs per egg mass, eggs per gram of fresh root (gfr), and reproduction factor (RF) were determined. Meloidogyne enterolobii infected and reproduced on all the sweetpotato cultivars. The nematode induced galls on both fibrous and storage roots, regardless of the cultivar, as well as induced necrosis and cracks on storage roots. The lesions and cracks on the storage roots were more visually pronounced on Hernandez than those on other cultivars. Cultivar Orleans sustained less root galling and egg masses than other cultivars (p ≤ 0.01), and both Orleans and Beauregard cultivars had less eggs per gfr and a lower RF than Covington (5,683 eggs/gfr; RF = 16.92), Evangeline (7,161 eggs/gfr; RF = 30.01), and Hernandez (6,979 eggs/gfr; RF = 22.6). The latter two cultivars sustained the largest amount of reproduction of M. enterolobii. The number of eggs per egg mass ranged from 462 to 503 and was similar among all cultivars. In summary, M. enterolobii reproduced well on all sweetpotato cultivars; however, differences were observed among cultivars (p ≤ 0.001). The host status as previously reported for other root-knot nematode species was not a good predictor of host status to M. enterolobii. Some sweetpotato cultivars that were reported as resistant or moderately resistant to M. incognita race 3, such as Evangeline and Hernandez, were among the best hosts to M. enterolobii. Root growth of Evangeline and Orleans, but not of the other cultivars, was negatively correlated with nematode eggs per gfr.The ability of Meloidogyne enterolobii to reproduce on selected sweetpotato (Ipomoea batatas) cultivars (Beauregard, Covington, Evangeline, Hernandez, and Orleans (LA 05-111)) was evaluated in two greenhouse experiments, each with 10 replicates. All cultivars, except Beauregard (control) and Orleans, were reported previously as moderately resistant or resistant to M. incognita, Fusarium oxysporum f. sp. batatas, and Streptomyces ipomoeae. Plants were inoculated with M. enterolobii (5,000 eggs/plant) and arranged in a completely randomized design in a greenhouse with an average daily temperature of 24.8°C. Galls and egg masses per root system (0-5 scale), eggs per egg mass, eggs per gram of fresh root (gfr), and reproduction factor (RF) were determined. Meloidogyne enterolobii infected and reproduced on all the sweetpotato cultivars. The nematode induced galls on both fibrous and storage roots, regardless of the cultivar, as well as induced necrosis and cracks on storage roots. The lesions and cracks on the storage roots were more visually pronounced on Hernandez than those on other cultivars. Cultivar Orleans sustained less root galling and egg masses than other cultivars (p ≤ 0.01), and both Orleans and Beauregard cultivars had less eggs per gfr and a lower RF than Covington (5,683 eggs/gfr; RF = 16.92), Evangeline (7,161 eggs/gfr; RF = 30.01), and Hernandez (6,979 eggs/gfr; RF = 22.6). The latter two cultivars sustained the largest amount of reproduction of M. enterolobii. The number of eggs per egg mass ranged from 462 to 503 and was similar among all cultivars. In summary, M. enterolobii reproduced well on all sweetpotato cultivars; however, differences were observed among cultivars (p ≤ 0.001). The host status as previously reported for other root-knot nematode species was not a good predictor of host status to M. enterolobii. Some sweetpotato cultivars that were reported as resistant or moderately resistant to M. incognita race 3, such as Evangeline and Hernandez, were among the best hosts to M. enterolobii. Root growth of Evangeline and Orleans, but not of the other cultivars, was negatively correlated with nematode eggs per gfr.

Morphological and Molecular Identification of Two Florida Populations of Foliar Nematodes (Aphelenchoides spp.) Isolated From Strawberry With the Description of Aphelenchoides pseudogoodeyi sp. n. (Nematoda: Aphelenchoididae) and Notes on Their Bionomics.

Two Florida populations of foliar nematodes were collected from strawberries originating from Cashiers, North Carolina (USA) located west from Willard, the type locality of Aphelenchoides besseyi. Both nematodes were cultured on Monilinia fructicola and identified using morphological characteristics and molecular assays as Aphelenchoides besseyi and Aphelenchoides pseudogoodeyi sp. n., a herein described new species related to Aphelenchoides goodeyi belonging to the Group of Aphelenchoides exhibiting stellate tails. The morphological and biological characters of Florida A. besseyi fit those of the original description of this species. A. pseudogoodeyi sp. n., which was initially misidentified as Aphelenchoides fujianensis, differed from the type population of the latter species from China because it was without males, and females lacked a functional spermatheca, whereas type A. fujianensis is an amphimictic species. Phylogenetic analyses using near full-length 18S ribosomal RNA (rRNA), the D2-D3 expansion fragments of 28S rRNA, and partial COI gene sequences indicated that A. besseyi is a species complex. A. pseudogoodeyi sp. n. grouped in different clades from those of the type A. fujianensis, instead merging with populations identified of 'A. fujianensis' from Brazil and other countries, suggesting that the latter are conspecific and incorrectly identified. The Florida A. besseyi infected strawberry and gerbera daisy, but not soybean and alfalfa. A. pseudogoodeyi sp. n. is mainly mycetophagous. Localized inoculation of 300 specimens applied with filter paper adhering to the blade of the soybean leaves resulted in nematode penetration into the mesophyll with subsequent development of lesions limited to the inoculated area of the blade.

Susceptibility of Seven Caladium (Caladium × hortulanum) Cultivars to Meloidogyne arenaria, M. enterolobii, M. floridensis, M. incognita, and M. javanica.

There is no known root-knot nematode (Meloidogyne spp.) resistance in caladium (Caladium × hortulanum), an ornamental foliage crop grown from tubers, but cultivars have been reported to differ in their level of susceptibility. Research was conducted to assess the relative susceptibility of seven widely grown caladium cultivars to the species of Meloidogyne which occur in the southeastern United States, where caladium cultivars are commonly planted in commercial and residential landscapes. Root-knot nematode species tested were Meloidogyne arenaria, Meloidogyne enterolobii (=M. mayaguensis), Meloidogyne floridensis, Meloidogyne incognita, and Meloidogyne javanica. All of the caladium cultivars tested were susceptible to galling by all species of Meloidogyne tested; however M. javanica caused the least severe galling. Meloidogyne enterolobii produced high numbers of eggs per gram of fresh root on all cultivars tested, with cv. Freida Hemple having the highest number (14,799 eggs/g fresh root). Meloidogyne javanica also reproduced at a high level on most cultivars tested. Overall, the number of eggs of M. arenaria, M. floridensis, and M. incognita was low on all caladium cultivars tested. Meloidogyne javanica was isolated from caladium roots in high numbers regardless of the cultivar. Meloidogyne incognita had low numbers of second stage root-knot nematode juveniles (J2) isolated from soil of all cultivars. The high level of reproduction of M. enterolobii and the high rate of isolation of M. javanica from roots, as well as the low rate of isolation of M. incognita from soil, are not reflected in gall ratings where M. javanica ratings were low but high numbers of eggs and J2 were present in roots. An increased understanding of cultivar susceptibility levels and the reproductive capacity of common root-knot nematode on caladium under various environmental conditions is needed to better manage nematode-infested planting sites and improve caladium growth.

Vertical Distribution of Pasteuria penetrans Parasitizing Meloidogyne incognita on Pittosporum tobira in Florida.

Pasteuria penetrans is considered as the primary agent responsible for soil suppressiveness to root-knot nematodes widely distributed in many agricultural fields. A preliminary survey on a Pittosporum tobira field where the grower had experienced a continuous decline in productivity caused by Meloidogyne incognita showed that the nematode was infected with Pasteuria penetrans. For effective control of the nematode, the bacterium and the host must coexist in the same root zone. The vertical distribution of Pasteuria penetrans and its relationship with the nematode host in the soil was investigated to identify (i) the vertical distribution of P. penetrans endospores in an irrigated P. tobira field and (ii) the relationship among P. penetrans endospore density, M. incognita J2 population density, and host plant root distribution over time. Soil bioassays revealed that endospore density was greater in the upper 18 cm of the top soil compared with the underlying depths. A correlation analysis showed that the endospore density was positively related to the J2 population density and host plant root distribution. Thus, the vertical distribution of P. penetrans was largely dependent on its nematode host which in turn was determined by the distribution of the host plant roots. The Pasteuria was predominant mostly in the upper layers of the soil where their nematode host and the plant host roots are abundant, a factor which may be a critical consideration when using P. penetrans as a nematode biological control agent.

Mitochondrial Haplotype-based Identification of Root-knot Nematodes (Meloidogyne spp.) on Cut Foliage Crops in Florida.

Florida accounts for more than 75% of the national cut foliage production. Unfortunately, root-knot nematodes (RKN) (Meloidogyne spp.) are a serious problem on these crops, rendering many farms unproductive. Currently, information on the Meloidogyne spp. occurring on most commonly cultivated cut foliage crops in Florida, and tools for their rapid identification are lacking. The objectives of this study were to (i) identify specific RKN infecting common ornamental cut foliage crops in Florida and (ii) evaluate the feasibility of using the mtDNA haplotype as a molecular diagnostic tool for rapid identification of large samples of RKN. A total of 200 Meloidogyne females were collected from cut foliage plant roots. Meloidogyne spp. were identified by PCR and RFLP of mitochondrial DNA. PCR and RFLP of mitochondrial DNA were effective in discriminating the Meloidogyne spp. present. Meloidogyne incognita is the most dominant RKN on cut foliage crops in Florida and must be a high target for making management decisions. Other Meloidogyne spp. identified include M. javanica, M. hapla, Meloidogyne sp. 1, and Meloidogyne sp. 2. The results for this study demonstrate the usefulness of the mtDNA haplotype-based designation as a valuable molecular tool for identification of Meloidogyne spp.

Multigene phylogeny of root-knot nematodes and molecular characterization of Meloidogyne nataliei Golden, Rose & Bird, 1981 (Nematoda: Tylenchida).

The root-knot nematodes of the genus Meloidogyne are highly adapted, obligate plant parasites, consisting of nearly one hundred valid species, and are considered the most economically important group of plant-parasitic nematodes. Six Meloidogyne species: M. arenaria, M. hapla, M. incognita, M. microtyla, M. naasi and M. nataliei were previously reported in Michigan, USA. For this study, Meloidogyne nataliei was isolated from the grapevine Vitis labrusca from the type locality in Michigan, USA, and was characterized using isozyme analysis and ribosomal and mitochondrial gene sequences. No malate dehydrogenase activity was detected using macerate of one, five, six, seven or ten females of M. nataliei per well. However, one strong band (EST = S1; Rm: 27.4) of esterase activity was detected when using homogenates of ten egg-laying females per well. Phylogenetic analyses of sequences of the partial 18S ribosomal RNA, D2-D3 of 28S rRNA, internal transcribed spacer of rRNA, mitochondrial cytochrome oxidase subunit I genes and the cytochrome oxidase subunit II-16S rRNA intergeneric fragment from fifty-five valid Meloidogyne species and M. nataliei were conducted using Bayesian inference and maximum likelihood methods. From these results, we infer 11 distinct clades among studied species, with M. nataliei and M. indica composing a basal lineage. Seventy five percent of these species belong to seven clades within the Meloidogyne superclade. Characterization of these clades is provided and evolutionary trends within the root-knot nematodes are discussed.