Morphological and molecular characterization, pathogenicity and co-infection effects of Ustilago kamerunensis with “candidatus phytoplasma oryzae” strain mbita 1 on napier grass growth tolerance under varying nutrients and moisture levels

Abstract

ABSTRACT: Napier grass (Pennisetum purpureum Schum.) is a leading feed for dairy animals in Eastern and Central Africa. In recent years the napier grass production has been threatened by napier head smut (NHS) and napier stunt disease (NSD), caused by Ustilago kamerunensis and “Candidatus Phytoplasma oryzae” strain Mbita 1 respectively. In efforts to manage the two diseases, host plant tolerance has been sought separately for each disease by selection of some tolerant accessions to napier stunt and head smut diseases respectively. However, there is no information available about the selected napier accessions’ ability in co-resisting the two diseases amidst reports of their continual spread in Kenya. Further, there is little understanding of the morphological and molecular characteristics of U. kamerunensis and its impact on the growth of the napier grass under co-infection treatments in varying nutrients and moisture levels. Thus, it is important to find out the real pathogen variations and their pathogenicity levels from the affected areas to inform the management strategies. The little knowledge available on molecular characteristics of U. kamerunensis pathogen has limited the extent of tolerant varieties adoption in different zones due to tolerance breakdown by the hypothesized isolates' varying pathogenicity levels. Therefore, the purpose of this study was to characterize U. kamerunensis isolates from the six affected counties in Kenya (Nakuru, Nyandarau, Kiambu, Kirinyaga, Murang’a and Nyeri), using the internal transcribed spacer 1, 2 and 5.8S rRNA genome elements. Further, evaluate their pathogenicity levels against napier grass growth tolerance under co-infection with “Candidatus Phytoplasma oryzae” strain Mbita 1 in varying nutrients and moisture levels. The U. kamerunensis propagules were systematically collected from the affected counties using geographic positioning system’s coordinates. This was done by dusting their ustilospores into pollination bags. Samples were transported to the laboratory for morphological and molecular analysis. Characterization involved in-vitro culturing of the isolates, colony colour observation and measurement of colony diameter. Total DNA of U. kamerunensis isolates was extracted and sequenced to determine the molecular variations. The two purposively selected U. kamerunensis (NAK002 & NYA002) isolates based on their growth in-vitro were studied in confined glasshouse conditions under co-infection of napier varieties (KK1, KK2, 16789 & Bana) with “Candidatus Phytoplasma oryzae” under four nutrient formulations (complete nutrient solution, nitrogen deficiency, phosphorus deficiency & nitrogen-phosphorus deficiency) and two watering regimes ( daily & weekly) in a completely randomized design experiment at ICIPE-Mbita. Growth parameters measured were; tiller height, tiller & leaf number, leaf area, chlorophyll content levels, total fresh, total stem and total leaf weights. Statistical analyses were conducted at confidence level of P ≤ 0.05. The study revealed differences in U. kamerunensis isolates based on morphological and molecular characteristics. Kiambu, Nyandarau and Nakuru isolates clustered together, as well as Murang’a, Nyeri and Kirinyaga. Further, differences were observed on their pathogenic levels. The sole NSD (“Candidatus Phytoplasma oryzae”) pathogen and NAK002 isolate infections were the most and least virulent respectively in comparison to co-infected treatments. The growth and tolerance levels of the evaluated varieties against the pathogens differed significantly. The varieties under nitrogen formulations and watering daily had high levels of pathogen tolerance. The ability of the varieties to maintain growth, chlorophyll stability and disease tolerance can be used in selecting highly tolerant germplasm. These findings would help farmers, plant pathologists and breeders to integrate information on molecular characterization, pathogenicity levels, varieties growth and tolerance levels under co-infection in streamlining the diseases management towards improving productivity and breeding aspects of Napier grass