Characterization of wheat nematodes from cultivars in South Africa
Lamula, Siphamandla Qhubekani Njabuliso
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Plant-parasitic nematodes (PPNs) naturally live in soil and attack small roots, but some species inhabit and feed in bulbs, buds, stems, leaves, or flowers. This leads to plant weakness and they often appear to suffer from drought, excessive soil moisture, sunburn or frost, as well as mineral deficiency or imbalance. According to the South African Plant-Parasitic Nematode Survey (SPPNS) and National Collection Nematodes (NCN) databases, 453 plant-feeding nematodes have been recorded in South Africa and species identified from wheat have not been included on the database as a result of not being georeferenced and the need to update information on NCN database. The aim of the current study is to characterize and document wheat nematodes that occur in South African wheat producing areas with an emphasis on morphological and molecular identification. A total of 776 composite rhizosphere soil and root samples was collected from 56 localities over two seasons in 7 provinces; the Free State (FS), KwaZulu-Natal (KZN), Northern Cape (NC), Mpumalanga (MP), Limpopo (L), North West (NW) and Western Cape (WC) provinces. The majority of samples collected were in WC (452), FS (120) and NC (160), as these are the major producers of wheat in South Africa. Nematodes were extracted from soil and root samples by modified decanting and sieving baermann-funnel technique, followed by the adapted sugar centrifugal-floatation. Nematodes were extracted from kernels by means of soaking the samples in tap water for 24 hours and decanting the extract through a 20 μm sieve. Nematode species were identified basis on morphological features, while prominence values (PV), frequency of occurrences and abundances were calculated for each genus. Individuals from the following genera were identified from the following provinces: Free State: Pratylenchus, Spiral (Rotylenchus, Scutellonema and Helicotylenchus), Criconema and Dolichodorus; KwaZulu-Natal: Melodogyne, Pratylenchus, Criconema, Helycotylenchus (Spiral) and Longidorus; Northern Cape: Pratylenchus, Criconema and Spiral; Western Cape: Pratylenchus, Rotylenchus, Scutellonema, Helicotylenchus, Coslenchus, Tylenchus and Xiphinema. The predominant genera identified across the localities according to PV-value were Pratylenchus (PV=575), Spiral (PV=309), Tylenchus (PV=348), Criconema (PV=377), Aphelenchus (PV=131) and Xiphinema (PV=32). Cedara in KwaZulu-Natal had a high number of Meloidogyne (420 second-stage juveniles /200 g soil) compared to the other sampled localities. Western Cape (Malmesbury) showed a high number of Pratylenchus sp. (8750/200 g soil). In root samples, the PV of Meloidogyne in Cedara was 183 followed by De Vlei (PV=943), Tygerhoek (PV=490), Kopporfontein (PV=134) and Wellington (PV=57) for Pratylenchus sp. No plant-parasitic nematodes were found in kernel samples. However, a bacterivore species of Panagrolaimus was identified from wheat kernels from Clarens in the Free State, is first record both in South Africa and worldwide. DNA was extracted from 320 nematodes using chilex method. PCR was used for amplification of 18S rRNA, ITS1, D2-D3 and CO1 genes and positive PCR products were sequenced. This study analysed 20 sequences of the D2–D3 expansion segments of the 28S rRNA gene, 80 sequences of the 18S rRNA and 91 sequences of ITS1 gene. Gene sequences of nematodes obtained from this study matched with their related species when subject to BLASTn on NCBI data. Phylogenetic trees constructed with 18S rRNA and ITS1 rDNA genes have shown that nematodes detected in this study including Helicotylenchus dihystera, Amplimerlinius paraglobigerus, Bitylenchus maximus, Merlinius joctus, Paralongidorus bikanerensis, Hoplolaimus galeatus and Rotylenchus unisexus each formed clades with strains of the same species obtained from the GenBank using the 18S rRNA gene. The H, dihystera, H. pseudorobustus, R. brevicaudatus, B. ventrosignatus and Mesocriconema sphaerocephalum also clustered with the strains of the same species obtained in the GenBank when using the ITS1 gene clusters with their related species from other countries.