Morphological and Molecular Characterization of Two Aphelenchoides Endophytic in Poplar Leaves

Abstract

During a long-term, large network study of the ecology of plant endophytes in native habitats, various nematodes have been found. Two poplar species, Populus angustifolia (narrowleaf cottonwood) and Populus trichocarpa (black cottonwood), are important ecological and genomic models now used in ongoing plant-pathogen-endophyte interaction studies. In this study, two different aphelenchid nematodes within surface-sterilized healthy leaves of these two Populus spp. in northwestern North America were discovered. Nematodes were identified and characterized microscopically and molecularly with 28S ribosomal RNA (rRNA) and 18S rRNA molecular markers. From P. angustifolia, Aphelenchoides saprophilus was inferred to be closest to another population of A. saprophilus among sequenced taxa in the 18S tree. From P. trichocarpa, Laimaphelenchus heidelbergi had a 28S sequence only 1 bp different from that of a Portuguese population, and 1 bp different from the original Australian type population. The 28S and 18S rRNA trees of Aphelenchoides and Laimaphelenchus species indicated L. heidelbergi failed to cluster with three other Laimaphelenchus species, including the type species of the genus. Therefore, we support a conservative definition of the genus Laimaphelenchus, and consider these populations to belong to Aphelenchoides, amended as Aphelenchoides heidelbergi n. comb. This is the first report of these nematode species from within aboveground leaves. The presence of these fungal-feeding nematodes can affect the balance of endophytic fungi, which are important determinants of plant health.

Keywords: nematode ecology; phylogeny; ribosomal DNA; systematics; taxonomy.

Fig. 1

Aphelenchoides cf. saprophilus male. A. Body, lateral view. B. Pharynx with stylet (S), median bulb, and pharyngeal gland (G). C. Tail with spicule. D. Tail tip with mucro, endophytic from within healthy, surface-sterilized narrow leaf cottonwood leaves, Populus angustifolia.

Fig. 2

Laimaphelenchus heidelbergi/Aphelenchoides heidelbergi n. comb. female from within healthy, surface-sterilized black cottonwood leaves, Populus trichocarpa. A. Body, lateral view with vulva (arrow). B. Vulva, ventral view (arrow). C. Offset tail tip with single tubercle.

Fig. 3

Bayesian phylogenetic tree of Aphelenchoides and Laimaphelenchus species inferred from an 806-bp fragment of 28S ribosomal DNA (rDNA). Sequences were aligned in ClustalW. In analysis, Markov chain was run for 1,100,000 generations, with first 100,000 trees discarded as burn-in. LnL mean/run = −4184.6. Node support is given above branches to left of nodes. Sequence generated in this work is indicated by an arrow.

Fig. 4

Bayesian phylogenetic tree of Aphelenchoides and Laimaphelenchus species inferred from an 1846-bp fragment of 18S rDNA. Sequences were aligned in ClustalW. Sequences were analyzed under a Tamura–Nei model. Markov chain was run for 1,100,000 generations, with first 100,000 trees discarded as burn-in. LnL mean/run = −13413.144, −13413.594. Node support is given above branches to left of nodes. Sequences generated in this work are indicated by arrows.