First Report of Meloidogyne javanica Infecting American Chestnut Trees (Castanea dentata) in Georgia, U.S.A

The American chestnut (Castanea dentata) is a monoecious deciduous tree in the beech family. It played a vital role in the ecosystem and industrially as a food source, providing rot-resistant wood and tannin for tanning leather. In the summer of 2018, suspected nematode-infected roots of American chestnut were collected from a backyard tree in Tift County, Georgia. Washing the roots with water and examining them under a dissecting microscope revealed many small galls and nematode females. A total of 45 root-knot (Meloidogyne spp.) and 22 ring (Mesocriconema spp.) nematodes were recovered per 100 cm³ of infested soil using a centrifugation flotation technique. Initial identification of the Meloidogyne specimens was performed by assessing morphological characteristics of the second-stage juveniles (n = 10) and perineal pattern of females (n = 4) as described in Eisenback and Triantaphyllou (1991), and the nematode was diagnosed as M. javanica (Treub, 1885; Chitwood, 1949). To confirm the species identity, DNA-based analyses were conducted. A single female nematode (n = 3) was isolated and transferred into a 1.5-ml microcentrifuge tube for molecular analysis. DNA was extracted using an Extract-N-Amp Tissue PCR Kit (Sigma-Aldrich, St. Louis, MO) following kit protocol with some minor modifications. Initially, the nematode was identified based on the fragment between the mitochondrial COII gene and the large (16S) rRNA gene with forward C2F3 (5′-GGTCAATGTTCAGAAATTTGTGG-3′) and reverse 1108 (5-TACCTTTGACCAATCACGCT-3) primers (Powers and Harris 1993). A 1.7-kb fragment characteristic of M. javanica was amplified, confirming the identity of the causal species. For further confirmation, PCR was also performed by selecting a species-specific sequence characterized amplified region using a primer set composed of Fjav (5′-GGTGCGCGATTGAACTGAGC-3′) and Rjav (5′-CAGGCCCTTCAGTGGAACTATAC-3′). It produced a specific fragment of the expected size (∼670 bp) for M. javanica (Donkers-Venne et al. 2000). Sequence analysis revealed that the pathogen had 99% sequence homology with M. javanica (GenBank accession no. KF041322). The sequence from our isolate was deposited in GenBank under the accession number MK451957. Koch’s postulates were fulfilled to verify the pathogenicity of the isolated nematode from infected C. dentata roots. Pure American chestnut seedlings were grown in pots with a potting medium consisting of coarse vermiculite and Promix (1:1 ratio) in the greenhouse. Four plants were inoculated by adding 2,000 eggs/pot of M. javanica. Four noninoculated seedlings were maintained as control plants. The plants were kept in a greenhouse at 24 to 26°C. After 75 days, the roots of all inoculated plants exhibited slight galling similar to that observed in the homeowner’s garden. An average of 328 eggs/g of root were recovered from the infected roots. Reproduction factor of the nematode was 4.1, showing that American chestnut was a good host. No disease symptoms were observed on the control plants. There is a prior report of M. querciana (Golden 1979) and Meloidogyne sp. (Siddiqui et al. 1973) infecting American chestnut, but to the best of our knowledge, this is the first report for M. javanica. Our findings are of great importance to American chestnut tree growers to help them establish management measures for M. javanica and understand possible interactions with other soilborne pathogens of chestnut.