Comparative analysis of fungal biocontrol agents on Meloidogyne species infestation on potato cultivars under different rhizosphere amendments

Abstract

Potato (Solanum tuberosum L.) is a well-known food crop that is regarded as an important component in the worldwide battle against hunger and malnutrition. Potato has significant potential to help contribute to Africa's rising food demands since it can produce more food per hectare than most crops. However, among many other pests and diseases, root-knot nematodes (RKN), Meloidogyne species, are serious limitations in the potato industry. Yield reduction of potatoes is mainly due to tuber quality and quantity. Given the significant economic impact caused by RKN, a variety of nematode-control techniques, including the use of synthetic chemical nematicides, have been developed. The discontinuation of several pesticides as a result of the United Nations Framework Convention on Climate Change multilateral agreements has nonetheless increased the need to implement effective ecologically safe nematode management practices. Using bio-control strategies has been suggested as a considerably safer and more feasible option for managing RKN. Currently, a diverse array of species, including nematophagous fungi are known to serve as bio-control agents (BCA) against RKN. These eukaryotes have a selective adaptation to different soil and climatic conditions such as temperature, pH and salinity levels, which further influences their interaction with RKN and their host. Considering these factors, it may be inferred that indigenous fungal BCA would stand a better chance at establishment in the rhizosphere and managing target pathogens as compared to exotic species. The study focused on eight major areas, (1) Screen for M. enterolobii and M. javanica nematodes susceptibility of five potato cultivars, viz., 'Buffelspoort P1', 'Hertha', 'Larnoma', 'Mnandi' and 'Up-to-date'; (2) Isolateand identify indigenous fungal species as possible candidates for bio-control investigation; (3) Investigate parasitism of the indigenous fungal species on M. enterolobii and M. javanica eggs and juveniles in vitro; (4) Investigate pathogenesis of nematode-parasitic fungi on selected seeds, viz., tomato, beetroot, swiss chard, dry bean, maize, sorghum and pumpkin; (5) Determine growth rate and geotropism of fungal species on different grain substrates, viz., red sorghum, white sorghum, barley, wheat and wheat bran; (6) Carry out in vitro screening of fungal species tolerance to stress and (7) Determine the effect of fungal species on M. enterolobii and M. javanica population densities on susceptible potato cultivar with and without compost incorporation under net house and field conditions. Reliability of measured variables was attained by using statistical levels of significance (P ≤ 0.05) and coefficient of determination (R2), with validity being ensured by conducting experiments at the same location over two seasons. Results of the first objective consistently demonstrated that potato cultivars, i.e., 'Buffelspoort 1', 'Hertha', 'Larnoma', 'Mnandi' and 'Up-to-date' were hosts to M. enterolobii and M. javanica, with the test nematodes inflicting damage to plants. Quadratic relationships between nematode reproductive factor (RF) values and initial population (Pi), in addition to confirming the density-dependent growth patterns of plant-parasitic nematodes(PPN), confirmed that the five test potato cultivars were susceptible to the two Meloidogyne species. Using serial dilution, a total of 25 fungal biotypes were isolated from soils collected from the rhizosphere of various fruit trees and vegetable plots. Soil samples were collected from plots with no previous record of fungal inoculations at the Agricultural Research Farm,North-West University (NWU), Molelwane, Mmabatho, North-West Province (25°48´36˝S, 25°37´ 47.999˝E). The biotypes were morphologically identified to genus level using colony characteristics, i.e., conidia, hyphal form and conidiophores. Using the molecular identification \polymerase chain reaction (PCR) method, eight fungal biotypes were confirmed as follows: A.flavus (MT645322.1), A. terreus (MT316343.1), Penicillium sp. AL-38 IRH-2012b(KC341982.1), Talaromyces minioluteus (MN788118.1), Tal. purpureogenus (AB872818.1), Tal. sayulitensis (MZ014549.1), Tri. ghanense (MF078652.1) and Tri. viride (MW456070.1). In vitro fungal nematophagous assessment of the eight fungal species was carried out by assessing the efficacy of indigenous fungal species on Meloidogyne species' eggs + secondstage juveniles (J2s) at 24, 48, 72 and 96 hours after exposure. Talaromyces sayulitensis had the highest M. enterolobii eggs parasitism (100%), in comparison to A. terreus which had the lowest parasitism (28%). Aspergillus flavus, Tal. purpureogenus and Tal. sayulitensis had the highest parasitism on M. javanica eggs (100%). On the other hand, Penicillium sp. had the lowest parasitic effects (55%). Talaromyces sayulitensis had the highest parasitic effect on M. enterolobii J2s (95%) and A. terreus had the lowest parasitism (60%). Aspergillus flavus and Tal. sayulitensis had the highest parasitic effects on M. javanica J2s (95%), whereas Tri. ghanense had the lowest parasitism (61%). The trend of the results showed that parasitism of the test fungi on Meloidogyne eggs and J2s increased with increasing time of exposure. The in vitro results exhibited that the test fungal species have the potential to be applied as BCA against Meloidogyne nematodes. Given that certain fungal species are parasitic and therefore with the potential of destroying many crops; their pathogenic effects were further assessed on different crops' seeds of economic importance, viz., tomato, dry bean, swiss chard, beetroot, maize, sorghum and pumpkin. It was observed that species within the same genus could affect crops in both favourable and unfavourable conditions. For instance, whilst A. terreus improved seed germination and seedling growth, A. flavus caused seed rot and reduced seed germination. It was demonstrated that the screened indigenous nematophagous fungal species, except for A. flavus, have exhibited seed germination enhancement and seedling enhancement. For the fungal isolates to be commercially developed for use as bio-control agents for problematic pathogens they should easily be produced and bulked. To commercially produce these fungal inoculants, they need to be cultured and bulked in the laboratory. Different grain substrates, viz., red sorghum, white sorghum, barley, wheat and wheat bran were screened for their performance as growth media for these respective fungal species. The fastest growth was observed on red sorghum followed by barley, white sorghum and wheat; whereas wheat bran showed the least growth. On red sorghum, all fungal species grew towards both positive (+ve)and negative (-ve) geotropism, i.e., downwards and upwards, respectively. Trichoderma viride recorded the highest +ve growth at an average of 9.3 cm. On the other hand, Tal. minioluteus showed the highest negative (-ve) tropism growth at 8.8 cm. Although at varying degrees, all species exhibited the most rapid -ve geotropism growth on substrates, except for Tal. purpureogenus, which failed to grow at all on wheat bran. The grain substrate, especially the red and white sorghum, technique was effective in mass-producing the studied fungal species. Ideally, to successfully dominate and protect plants from stressors, BCA should tolerate varying environmental conditions when they are introduced. However, BCA are living microorganisms and their survival and efficacy can be impeded by extreme conditions. Accordingly, the study further evaluated whether indigenous nematophagous fungal species could tolerate different levels of salinity, pH, nutrients and temperature. The results showed that, although being indigenous, Aspergillus, Tal. sayulitensis and Tri. ghanense failed to thrive in saline conditions and high pH. Compared to other species, Penicillium sp. failed to thrive under reduced nutrient levels. All fungal species exhibited minimal to no growth at 10-20 °C. Five nematophagous fungal species that showed the highest in vitro parasitism on Meloidogyne eggs + J2s were further assessed for their ability to reduce M. enterolobii and M. javanica population density on potato cultivar 'Hertha' with and without the incorporation of organic compost (OC) in vivo. The in vivo experiments were under a net house and repeated under field conditions. Results showed, though inoculated with Meloidogyne species; plant height, chlorophyll content, fresh shoot weight, dry shoot weight and bulb weight were significantly increased when administered with nematophagous fungal species (P ≤ 0.05). On the other hand, gall rating, final J2s population density (Pf) and RF were significantly decreased (P ≤ 0.05). These findings imply that the efficacy of indigenous nematophagous fungal species can be improved by the application of OC to control Meloidogyne nematode infection and can be used as an environmentally safe alternative method to synthetic nematicides.