A morphological and molecular study of Heterodera carotae Jones, 1950 in South Africa
Carrot (Daucus carota L.), an important vegetable crop, is cultivated worldwide for the fresh market and processing industry. In South Africa carrot is one of the four major root and tuber vegetables consumed. The objectives of this study were to 1) determine the different life stages of Heterodera carotae Jones, 1950 on a weekly basis and the number of life cycles completed per growing season, and 2) characterize the carrot cyst nematode population from the Tarlton area, Gauteng Province (South Africa) morphologically and molecularly. No formal morphometric or molecular identifications have been performed on H. carotae for the African continent, neither have soil tolerance limits been determined. Tap root and corresponding rhizosphere samples were obtained from a one-hectare carrot trial block in the Tarlton area during the 2016-2017 summer growing season. This was done weekly for 18 weeks. Nematodes were extracted from the samples using the sieving centrifugal flotation and Seinhorst cyst elutriator methods, depending on the life stages required. Accumulation of day degrees, counting and identifying nematode of life stages was done to determine the number of life cycles and the length of the life cycle of the Tarlton population. Infective second-stage juveniles (J2), females, cysts and males were used for morphological and molecular identification using light microscopy (LM) and scanning electron microscopy (SEM). For molecular identification, deoxyribonucleic acid (DNA) extraction and the polymerase chain reaction (PCR) were used for the internal transcribed spacer (ITS)-rDNA region. Interestingly, J2 and young (yellow cysts) were present in as early as the first week and cysts during the second week of sampling. This may have been attributed to remnants of the previous carrot crop still persisting in the soil. Two life cycles of H. carotae were recorded for the 18-week study period. Morphological and morphometrical characterization of H. carotae, using LM and SEM, suggested the Tarlton population to be conspecific with H. carotae populations described from Europe and Canada. Observed differences between the morphometrics of various life stages of the Tarlton population lay within the range of intraspecific variation compared to that of the aforementioned, described H. carotae populations. Morphometrics and morphological identification revealed that J2 of the Tarlton population closely resembles H. carotae, except for 1) the slightly shorter body length of some specimens compared to that of the European and Canadian populations, 2) the smaller head diameter of the Tarlton J2 specimens compared to that of European populations, 3) slightly smaller cysts from Tarlton compared to those from European and Canadian populations, 4) shorter underbridge of cysts from Tarlton compared to that of European populations and 5) slightly longer spicules of males from Tarlton than those for both European and Canadian specimens. Nonetheless, the vulval slit length of Tarlton cyst specimens compared well with that of cysts from European and Canadian populations. Molecular results indicated a 99 % similarity of the Tarlton population to three H. carotae populations selected from the NCBI Genbank database. Based also on this molecular analysis, the Tarlton population is considered to be conspecific with described H. carotae populations. This study added novel and valuable information about a South African carrot cyst nematode population: a species that is of great economic importance for local producers.