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.

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.

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.

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.

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.