Parasitism effects on white clover by root-knot and cyst nematodes and molecular separation of Heterodera daverti from H. trifolii

This research carried out an accurate identification of the root-knot and cyst-forming nematode species parasitizing white clover at the Laceno Lake area in Southern Italy. Two species, the root-knot nematode Meloidogyne hapla and the cyst nematode Heterodera daverti were identified by integrative taxonomic approaches (classical, isozyme pattern, and D2-D3 expansion segments of 28S rRNA, ITS rRNA and coxI of mtDNA gene sequences) and found parasitizing white clover roots. These nematodes were detected in stunted plants with a reduced number of rhizobium nodules and the host suitability was confirmed by the high nematode population densities ranging from 53 to 2350 eggs and J2s per g of fresh roots for M. hapla, and 1.36 eggs and J2s/cm³ of soil for H. daverti; and cyst nematode females were also detected on the roots of clover. The studies on the host-parasite relationships of nematode-feeding sites in white clover roots infected by these nematodes showed a high susceptible response. Meloidogyne hapla and H. daverti infections were also observed on nitrogen-fixing root nodules of white clover, where well established feeding sites allowed active nematode reproduction. Histological examination of nitrogen-fixing root nodule tissues revealed that the nematodes established their permanent feeding sites in the vascular bundles of nodules which appeared enlarged deformed and disorganised by the expansion of nematode feeding cells (giant cells and syncytium) and hyperplasia of the nodule cortex. Additionally, coxI of mtDNA gene is an efficient barcoding sequence for discriminating the identification of H. daverti from H. trifolii.