Distribution, Characterization and Management of Papaya Ringspot Associated Viruses in Kenya Naomi Nzilani Mumo A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Horticulture of the Jomo Kenyatta University of Agriculture and Technology 2022

Abstract

Papaya (Carica papaya L.) is an important fruit crop in Kenya, grown by small and large-scale farmers for local and export markets, providing income to many papaya producers. Ripe fruits are rich in vitamins A and C, minerals and dietary fiber. Consumption of these fruits is important in preventing vitamins and mineral deficiency in developing countries including Kenya. Production of papaya, however, is constrained by papaya ringspot. Farmers’ knowledge, perceptions and management practices of the disease in the country is not documented. Further, the virus (es) associated with the disease as well as their incidence and distribution are not established. In addition, the host range of the virus associated with the disease in papaya is unknown. These information are important in designing long-term and sustainable papaya ringspot management strategies both at a community level and in the country. In the current investigations, farmers’ knowledge, perceptions, and management practices for the disease, were studied using a semi-structured questionnaire that was administered through face-to-face interviews to 103 smallholder farmers in five major papaya growing regions and 22 counties in Kenya. The results showed that 38.8 % identified the disease, with 48.8 % of those who identified the disease not knowing the cause. The disease was regarded as a moderate to serious constraint to papaya production by the majority of the respondents. As a management measure, slightly more than fifty percent (54.8 %) sprayed plants showing the disease symptoms with chemical insecticides, 4.7 % removed the infected plants from the field while 40.5 % did not apply any management measure on the diseased plants. It is therefore concluded that papaya farmers in the sampled regions have limited awareness and knowledge and management of papaya ringspot. It is therefore recommended that capacity building of the papaya farmers on proper identification and management techniques of the disease be undertaken. The viruses associated with disease were studied in 48 plant samples collected from the 22 counties subjected to Next-generation sequencing (NGS) using the Illumina MiSeq platform. Sequence analysis revealed the presence of Moroccan watermelon mosaic virus (MWMV), a potyvirus, and three Carlaviruses; Cowpea mild mottle virus (CpMMV), and two putative Carlaviruses, closely related to cucumber vein-clearing virus (CuVCV). In reference to typical symptoms observed in the infected plants and sequence similarities with CuVCV, the two putative Carlaviruses were named papaya mottle-associated virus (PaMV) and papaya mild mottle-associated virus (PaMMV). Disease incidence was determined from twenty plants in every field surveyed for papaya ringspot-like symptoms. The highest disease incidence of 71.4, 51.4 and 52.8 were reported in Kiambu, Murang’a and Nakuru counties respectively. The least incidence were recorded in Kwale (3.8) and Busia (2.8) counties. The papaya ringspot prevalence differed across the regions surveyed. A 100 % disease prevalence was reported in Elgeyo Marakwet, Embu, Homabay, Kiambu, Nakuru, Kitui and Vihiga counties. The disease severity was mild (with a severity index of 2.9) across the surveyed counties. Two hundred and eighty-seven leaf samples collected from farmers’ fields tested for MWMV, CpMMV and PaMV viral infections showed that MWMV was the most widespread with 140/287 samples testing positive from 11 counties. The PaMV was the second most prevalent virus detected in 39/287 and 9 of 22 counties. CpMMV was the least prevalent and was detected in 7/287 of samples collected and in three counties. Occurrences of MWMV and PaMV were detected in five counties; Embu, Kirinyaga, Meru, Machakos and Makueni while that of PaMV and CpMMV was detected in Baringo, Meru and Kitui Counties. The results showed that MWMV was associated with papaya ringspot in Kenya and is also widespread in the country. Other viruses previously not known to infect papaya were detected which could pose a threat to papaya production in the country. Therefore, screening papaya seedlings for the viruses before planting would be an important strategy in preventing disease spread. The host range of the MWMV infecting pumpkin intercropped with MWMV infected papaya plants was identified through NGS and compared. The MWMV isolate from papaya and pumpkin were sap inoculated onto 14 plant species belonging to four families; Datura metal, D. stramonium, Nicotiana clevalendii, N. tabacum, N. glutanosa, N. bentamiana, Cucurbita pepo (zucchini), C. moschata (pumpkin), Citrullus lanatus (watermelon), Cucumis sativus (cucumber), Phaseolus vulgaris (common bean), Vigna unguiculata (cowpea), V. radiate (mung bean) and Carica papaya L. in the greenhouse. From the results, the MWMV infecting pumpkin was a different strain from that infecting papaya; sharing 83.4-83.7 % nucleotides (92.3-95.1 % amino acids) sequence identities in coat protein. Through sap inoculation, MWMV isolated from pumpkin infected watermelon, cucumber and zucchini, but unable to infect papaya and other plant species tested. Similarly, MWMV infecting papaya infected zucchini but did not infect other plant species tested suggesting the existence of independent strains having different molecular and biological characteristics associated with the host specificity. Further research should focus on determining transmission of the viruses using vectors.