Report of the Parana coffee root-knot nematode, Meloidogyne paranaensis (Tylenchida: Meloidogynidae) from Caladium sp. in the continental United States

Abstract In May 2021, the Parana coffee root-knot nematode, Meloidogyne paranaensis was identified using molecular markers from a potted elephant ear plant (Caladium sp.) originated from San Antonio, Texas, USA. This nematode was found in a mixture with the peanut root-knot nematode, Meloidogyne arenaria. The molecular analysis showed that the intergenic COII-16S gene region and the D2–D3 of 28S rRNA gene sequences allowed differentiating M. paranaensis from the related root-knot nematode species of the tropical group. To the best of our knowledge, it is the first report of M. paranaensis in the continental United States.

In May 2021, the soil sample taken from a potted elephant ear plant (Caladium sp.) originating from San Antonio, Texas, USA was sent for analysis in the Nematology Laboratory, Plant Pest Diagnostics Center, California Department of Food and Agriculture, Sacramento, California. Several second-stage juveniles (J2) of the root-knot nematodes (RKN) were detected in the extracts from this soil sample. The analysis of these juveniles using several molecular markers revealed that this sample contained a mixture of two root-knot nematode species: the Parana coffee rootknot nematode, Meloidogyne paranaensis (Carneiro et al., 1996) and the peanut root-knot nematode, Meloidogyne arenaria (Neal, 1889) Chitwood, 1949 To the best of our knowledge, it is the first report of M. paranaensis in the continental United States.
Meloidogyne paranaensis was first described in 1996 in the state of Paraná, Brazil (Carneiro et al., 1996;Campos and Vallain, 2005). This species is considered as one of the most destructive RKN species parasitizing coffee in Brazil and in the Americas. It has been also reported from Colombia, Costa Rica, Guatemala, and Martinique (Subbotin et al., 2021) as well as in Mexico (López-Lima et al., 2015) and Hawaii, USA (Carneiro et al., 2004).
The objective of the present study was to provide molecular characterization of M. paranaensis associated with an elephant ear plant (Caladium sp.).

Nematode extraction and morphological examination
Nematodes were extracted using the Baermann funnel method from the soil sample taken from a potted elephant ear plant (Caladium sp.) originated from Texas, San Antonio. Several second-stage juveniles (J2) killed by heating were morphologically examined and photographed using an automatic Infinity 2 camera attached to a compound Olympus BX51 microscope equipped with Nomarski interference contrast.
The new sequences for each gene were aligned using ClustalX 1.83 with their corresponding published gene sequences of M. paranaensis and other RKN species from the tropical group (De Ley et al., 2005;Tigano et al., 2006;Humphreys-Pereira et al., 2014;Alvarez-Ortega et al., 2019;Santos et al., 2020;and others). The alignment for the COII-16S gene sequences were used to construct phylogenetic networks using statistical parsimony (SP) as implemented in POPART software (http://popart.otago.ac.nz) (Bandelt et al., 1999).

Morphological study
A few J2s were extracted from the soil sample from potted Caladium plant. Photos of anterior and posterior   (Subbotin et al., 2021).

Molecular characterization
The intergenic COII-16S mitochondrial gene region Two new identical sequences were obtained in this study for M. paranaensis. The alignment was 604 bp in a length and contained 72 sequences of Meloidogyne species. The partial alignment is given in Fig. 2. The sequences of M. paranaensis contained two long deletion fragments (46 bp and 21 bp). The phylogenetic relationships of sequences of M. paranaensis with other the root-knot nematodes from the tropical complex reconstructed using SP are given in Fig. 3. Sequences of M. paranaensis from the USA were different from those from Brazil in 0.4% (2 bp).

The D2-D3 of 28S rRNA gene
Three new identical sequences were obtained in this study for M. paranaensis. Search of the D2-D3 of 28S rRNA gene sequences of M. paranaensis with Blastn in the Genbank showed 100% similarity (100% coverage) with 28S rRNA gene sequences of M. paranaensis (KY911101, KF993620, AF43800, AF435798) from Brazil. The alignment was 535 bp in a length and contained 112 sequences of Meloidogyne species. The phylogenetic relationships of sequences of M. paranaensis with other the root-knot nematodes from the tropical complex reconstructed using SP are given in Fig. 4.
The partial nad5 gene

PCR with specific primer
Conventional PCR with the M. paranaensis species specific primers designed by Randig et al. (2002) did not generate any amplicons with DNA sample of M. paranaensis obtained from Caladium plant (data not shown).

Discussion
The Parana coffee root-knot nematode is the most damaging species in Brazilian coffee plantations, where losses may reach 50% of the coffee yield (Carneiro et al., 1996). Although this nematode species is highly aggressive to Coffea arabica L., which is the primary host of this species, M. paranaensis has also been detected in tobacco, tomato, watermelon, several weeds and other plants (Carneiro et al., 1996;Mônaco et al., 2011;Subbotin et al., 2021;Tomaz et al., 2021). In this research we discovered Caladium sp. as a host plant for this nematode. Although, the plant infected with the Parana coffee root-knot nematode came from San Antonio, Texas, we cannot exclude that plant materials may have originally came from South and Central America, where Caladium plants, tropical perennials with colorful, heart-shaped leaves, are native to tropical forests.
Within the USA, the state of Hawaii grows their own coffee, however, California has recently established the coffee plantations in Santa Barbara and San Diego counties. Preventing the distribution of coffee pests to California will become an important task for diagnostic laboratories. Simply using the perineal pattern, J2s measurements and differential host tests could misidentify M. paranaensis as M. incognita, that has happened for years. Although analysis of rRNA and mtDNA gene sequences allows reliably identifying RKN species, new rapid, and cheap molecular diagnostic tools are needed for detection of the Parana coffee root-knot nematode.