Morphological and Molecular Analysis of Two Mycophagous Nematodes, Aphelenchoides bicaudatus and A. rutgersi (Nematoda: Aphelenchoididae) from Florida Strawberry.

From 2016 to 2021, nematode surveys in Florida strawberry fields revealed several species of foliar nematodes (Aphelenchoides spp.). Aphelenchoides besseyi sensu stricto was detected only in 2016 and 2017 on photosynthetic strawberry leaves/buds, but other not well characterized populations of Aphelenchoides sp. were found on declining/dessicated leaves. Morphological analyses showed that these samples of Aphelenchoides sp. consisted of A. bicaudatus, a species detected in Florida for the first time, and A. rutgersi, a species previously reported in Florida from the citrus rhizosphere. These two species differed from A. besseyi in the shape of their tail terminus: bifurcate in A. bicaudatus; mucronate with a ventral thin mucro in A. rutgersi; and stellate in A. besseyi. One population each of these species was used for morphological and molecular analyses after being reared on Monilinia fructicola. Body and tail length differences were observed among Florida A. bicaudatus and other populations from the Far East and South Africa. Phylogenetic analyses of the rRNA gene sequences showed that Florida A. bicaudatus grouped with those of species from South Korea, Taiwan, and the Netherlands and several other populations listed as Aphelenchoides sp. from Brazil, Costa Rica, and Japan, which were considered as representatives of A. bicaudatus in this study. Similarly, sequences of Florida A. rutgersi grouped with those from environmental samples in Japan and North Carolina, which were listed as Aphelenchoides sp. and were considered as representatives of A. rutgersi in this study. Photosynthetic strawberry leaf samples were free from both A. bicaudatus and A. rutgersi, indicating that these two species did not damage strawberry. They were associated with desiccated leaves and/or propagative stolons, usually infected by fungi, confirming that they are mycetophagous under field conditions in this study. Results of soybean leaf inoculation on moist filter paper containing A. bicaudatus specimens showed that this species could become phytophagous under artificial conditions. Nematodes penetrated the leaf epidermis and migrated into the mesophyll causing leaf tissue discoloration/necrosis, which remained localized within the infested area. Soybean leaf damage was almost negligible, and no nematode reproduction was observed in the inoculated soybean areas.

Detection of Resistance, Susceptibility, Tolerance, and Virulence in Plant-Nematode Interactions: Part I-Sedentary Endoparasitic Nematodes.

The use of nonhost, tolerant, or resistant plants, to manage plant parasitic nematodes (PPNs), is an appealing, economic, and environmentally friendly agronomic practice, which is effective when precise information on the identification of PPN species and their virulence to target host crops is available. This chapter describes suggested protocols to evaluate the reaction of the most important crops and fruit trees to infestation by the most damaging PPN with sedentary endoparasitic habits, with the aim of assessing resistance and tolerance traits, sources of resistance in progenies from breeding programs, the reaction to nematodes of newly released cultivars, and the virulence of the most noxious PPNs. These protocols consist of classical screening techniques not involving biochemical and molecular analyses. PPN species and genera considered in this chapter include (i) the most important species of root-knot nematodes Meloidogyne spp., including also M. chitwoodi, M. enterolobii, and M. graminicola, and (ii) the cyst-forming nematodes of the genera Globodera and Heterodera, such as the potato cyst nematodes (PCNs) Globodera rostochiensis and G. pallida, and also Heterodera avenae group, H. ciceri, H. glycines, and H. schachtii. Schemes are given to identify virulence groups for most of these nematodes.

Morphological and Molecular Diversity among Pin Nematodes of the Genus Paratylenchus (Nematoda: Paratylenchidae) from Florida and Other Localities and Molecular Phylogeny of the Genus.

Pin nematodes (Paratylenchus spp.) are root parasites found worldwide. They have different life cycles and feeding habits and can damage a wide range of plants. A remarkable diversity of pin nematode species was found in soil samples from Florida and other states of the USA, Canada, and Spain. Using integrative taxonomy, two new species (Paratylenchus hawaiiensis sp. n. and P. roboris sp. n.), six valid species (Paratylenchulus acti, P. aquaticus, P. goldeni, P. paralatescens, P. minutus (=P. shenzhenensis syn. n.), and P. straeleni), and two undescribed species were identified from Florida; P. goldeni, P. hamatus, P. hamicaudatus, P. holdemani, and P. pedrami were found in California, P. minutus in Hawaii, P. goldeni in Oregon and Washington, and one new species, Paratylenchus borealis sp. n., in Alaska. Outside the USA, Paratylenchus projectus was detected in samples from Canada and Spain as well as P. holdemani and Paratylenchus sp. from Spain. The pin nematode species from Belgium and Russia identified in former studies as Paratylenchus sp. F was herein described as a new species with the name of P. borealis sp. n., using a population from Alaska. Previously reported molecular type A of P. aquaticus from Hawaii was reclassified as P. hawaiiensis sp. n., using a population from Florida. Paratylenchus roboris sp. n. from Florida has obese sedentary females with a stylet 63-71 µm long. The results of the molecular analysis of P. shenzhenensis from Florida and China indicated that it was conspecific with P. minutus from Hawaii and considered here as its junior synonym. New 26 D2-D3 expansion segments of 28S rRNA, 17 ITS rRNA, and 20 COI gene sequences were obtained in this study. Phylogenetic relationships of Paratylenchus are reconstructed using the D2-D3 of 28S rRNA, ITS rRNA, and COI gene sequences. Congruence of molecular and morphological evolution and species identification problems are discussed. Obese females were found in two major clades of Paratylenchus. The problem of reference materials is discussed, and it is proposed to make more efforts to collect topotype materials of known Paratylenchus species for molecular study.

First Report of Direct Damage Caused by the Stubby-Root Nematode, Nanidorus minor, to Strawberry (Fragaria x ananassa), in Florida.

In 2019-2022, declining symptoms were observed in two commercial strawberry farms in Hillsborough County, Florida. The fields in the two farms consisted of raised beds covered by plastic mulch. Both were fumigated with a mixture of 1,3-dichloropropene (40%) + chloropicrin (60%) before planting. Samples collected from large patches with declining plants were infested with stubby-root nematodes. No sting and root-knot nematode species were detected. The results of morphological and molecular analyses indicated that the stubby-root nematode populations were representative of the species Nanidorus minor. The two cultivars 'Florida Brilliance' and 'Florida Sensation' in the two fields included plants with stubby root symptoms showing a reduction in the size of the root system and arrested growth and elongation of the feeder roots on the first strawberry crop. The nematode population densities in the two fields increased at the end of strawberry season and averaged 66 and 96 specimens in 200 cm3 soil. In one of the fields, a second strawberry crop was established as in the previous year using the same practices (fumigation and raised beds covered with plastic). However, in this field the population of N. minor declined and did not reach damaging levels at the end of the season on the second strawberry crop. The factors causing the decline of the nematode population were not elucidated. This is the first report of a direct damaging effect of N. minor to strawberry.

Morphological and molecular characterization of Xiphinemella esseri Chitwood, 1957 (Dorylaimida: Leptonchidae) from Florida, with the first molecular study of the genus.

A population of Xiphinemella esseri, recently collected under the canopy of associated live oak trees in north Florida, was studied and described with an integrative approach, including the first molecular study of the genus. This Florida population is characterized by its 2.30 to 3.32 mm long body, labial disc well developed, lip region offset by constriction, and 16.5 to 17.5 µm broad, odontostyle 46 to 49 µm long with minute aperture, neck 288 to 296 µm long, pharyngeal expansion occupying 28 to 30% of total neck length, uterus a tripartite tube-like structure, pars refringens vaginae absent, vulva transverse (V = 45.4-49.7%), tail short and rounded (18-28 µm, c = 94-158, c' = 0.6-0.9), spicules 41 to 45 µm long, and 8 to 10 irregularly spaced ventromedian supplements bearing hiatus. The phylogenetic analysis inferred from the D2-D3 expansion segments of 28S rRNA gene and 18S rRNA gene sequences showed that X. esseri clustered with other dorylaims from the family Leptonchidae. A brief discussion about the distribution and biological considerations of X. esseri is also provided.

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.