Isolation of Endophytic Beauveria bassiana and investigation of interactions between Fusarium spp., Eldana saccharina and the sugarcane plant for dual biological control = Ukwehlukanisa kwe-Beauveria bassiana enobukhunta kanye nokuphenya ubudlelwane phakathi kwe-Fusariuam spp., Eldana saccharina kanye nesitshalo somoba ukwengamela okuphilayo okuhamba ngakubuli.

Abstract

The South African Sugarcane Research Institute is exploring the use of the fungal entomopathogen Beauveria bassiana to control the borer pest Eldana saccharina, a major threat to the South African sugar industry. This study aimed to understand the natural occurrence of B. bassiana in various sugarcane genotypes and host plants of E. saccharina, and its interactions with the pest and a common plant co-occurring pathogen, Fusarium spp. which is beneficial to the pest. Therefore, this study aimed to survey the natural occurrence of B. bassiana as an endophyte in 28 sugarcane genotypes and E. saccharina host plants, to identify isolates that are virulent and can be used as endophytes in biological control programs to control the boring stages of E. saccharina and to explore the interaction of isolated B. bassiana isolates with Fusarium spp. Furthermore, this study investigated the persistence of B. bassiana as an endophyte in sugarcane plants and the ability of the endophytic association to reduce E. saccharina feeding. In this study, 128 fungal isolates morphologically characterized as Beauveria spp. were isolated as endophytes from internal plant tissues following the sampling of 326 plants collected from five locations in sugarcane-producing areas (KwaZulu-Natal, South Africa). Analysis of the nuclear ribosomal internal transcribed spacer (ITS) region confirmed the identity of 16 representative isolates as B. bassiana s.l., with ˃98% sequence similarity to reference strains. For the first time, 120 endophytic Beauveria spp. isolates were obtained from 22 sugarcane genotypes and eight from E. saccharina host plants. Sixteen B. bassiana isolates were repellent towards E. saccharina first instar larvae during larval choice assays, detrimental towards E. saccharina development when B. bassiana isolates were incorporated into E. saccharina artificial diets and virulent towards 3rd and 5th instar larval stages of E. saccharina during topical conidia spray bioassays. Interactions of two isolated B. bassiana with four Fusarium spp. strains revealed that both genera can reduce and limit the other’s growth when grown with culture filtrates or in Petri dish co-culture assays. Artificial inoculated B. bassiana isolates endophytically colonized sugarcane plants. For the first time, this study revealed that endophytic colonization can persist from 1 to 6 months in sugarcane with colonies resembling Beauveria spp. morphology re-isolated using selective agar. Endophytic colonization persistence decreased with an increase in time post inoculation, was found to be variety dependent and influenced by the inoculation method, plant pre-treatment, and fungal strain used. Sugarcane plants inoculation with B. bassiana TL-leaf strain via conidia spray inoculation, weighed significantly more than control plants 3 months post inoculation, with no statistical significance in plant height when compared to control plants. When E. saccharina larvae were allowed to feed on plants 16 months post B. bassiana inoculation, no statistically significant differences were observed within control and B. bassiana plants in reducing boring length and larval weights. Furthermore, no B. bassiana colonies were re- isolated 16 months post inoculation into sugarcane. The results of this study demonstrated the abundance of B. bassiana as an endophyte within plant species in the sugarcane ecosystem. It highlighted the importance of conducting bioassays when selecting biological control strains, as levels of repellence and virulence differ amongst B. bassiana strains. Additionally, the study revealed that using an appropriate B. bassiana biocontrol strain and artificial endophytic colonization of sugarcane is achievable in different sugarcane varieties. Moreover, results accentuate the importance of determining persistence of artificially inoculated B. bassiana strains as an endophyte in the plant ensuring B. bassiana is present as an endophyte in the plant when the target pest feeds.

Iqoqa. Isikhungo soCwaningo lukaMoba waseNingizimu Afrikha luphenya ukusetshenziswa kwesinambuzane esibanga ukhunta i-Beauveria bassiana ukulawula isinambuzane esidla umoba i-Eldana saccharina, okuyingozi enkulu emkhakheni kashukela eNingizimu Afrikha. Lolu cwaningo luhlose ukuqonda ukwenzeka okuvamile kwe-B. bassiana kulibofuzo lokuphilayo olwehlukene kanye nezitshalo ezamukelayo ze-E. saccharina, kanye nokuxhumana kwakho nesinambuzane kanye kanye negciwane elibanga ukhunta esitshalweni, i-Fusarium spp. Okunomvuzo ezinambuzaneni. Ngakho-ke, lolu cwaningo luhlose ukuhlola ukwenzeka kwemvela kwe-B. bassiana njengokhunta kulibofuzo lokuphilayo lukamoba olungama-28 kanye nezitshalo ezengamele ze-E. saccharina ukuthola izehlukaniso ezinobungozi futhi ezingasetshenziswa njengezinhlelo zokwengamela okuphilayo kokhunta ukwengamela izigaba ezingenamfutho ze-E. saccharina kanye nokuphenya ukuxhumana kwezehlukanisi ezizimele ze-B. bassiana kanye ne-Fusarium spp. Okunye futhi, lolu cwaningo luphenye ukubelesela kwe-B. bassiana njengokhunta ezitshalweni zomoba nokukwazi kwenhlanganisela yokhunya ukwehlisa ukuphikelela kwe-E. saccharin. Kulolu cwaningo, izehlukanisi zokhunta eziyi-128 ngokwakheka kwazo okubonakala nge-Beauveria spp. kwahlukaniswa njengolibofuzo lokuphilayo ezicutshini ezingaphathi zesitshalo ngokulandela ukusampula kwezitshalo ezingama-326 ezaqoqwa ezindaweni ezinhlanu zezindawo ezikhiqiza umoba (KwaZulu-Natali, eNingizimu Afrikha). Ukuhlaziya komongo wenhlanganisela yephrotheni nenhlayiya esifundeni se-internal transcribed spacer (ITS) kuveze ubukhona bezimeleli eziyi-16 zezahlukanisi njenge-B. bassiana s.l., ngohlelo olufanayo olu- ˃98% ukuhlobanisa nengcindezi. Okokuqala, ukhunta olu-120 lwezihlukanisi ze-Beauveria spp. lwatholakala kulibofuzo lokuphilayo olungama-22 lukamoba kanye nezitshalo ezengamela eziyisishiyagalombili ze-E. saccharina. Izehlukanisi eziyishumi nesithupha ze-B. bassiana zazivikela i-E. saccharina isigaba sokuqala sezibungu ngesikhathi zokuhlola uhlobo lwezibungu, okunobungozi ekuthuthukeni kwe-E. saccharina ngesikhathi izehlukanisi ze-B. bassiana zihlanganiswe nezakhi zokuzenzela ze-E. saccharina futhi ezinobungozi ezigabeni zokuqala ezingesesithathu nesesibili ze-E. saccharina ngesikhathi sokuhlola okuphilayo kokusabalala kokhunta lokuqala. Ukuxhumana kwezehlukanizi ezimbili ze-B. bassiana kanye nengcindezi ye-Fusarium spp. kwaveza ukuthi zozimbili zinhlobo zingehlisa futhi zinqinde ukukhula kokunye uma kukhuliswe nezinsalela zofuzo noma ekuhloleni ufuzo oluhambisanayo lwe-Petri dish. Ukujovwa okungekhona kwezehlukanisi ze-B. bassiana kukhuntise ngokuqonela izitshalo zomoba. Okokuqala, lolu cwaningo luveze ukuthi ukukhuntisa ngokuqonela kungadlondlobala kusukela kwe-1 kuya kweziyi-6 izinyanga emobeni ngemiqoneli efuze ukwakheka kwe-Beauveria spp. okuphinde kwehlukaniseke ngokusebenzisa uketshezi olujiyisa ufuzo olukhethekile. Ukuphikelela kokuqonela kokhunta kwehla kwenyuka ngesikhathi esingemumva komjovo, kwatholakala kwencike ngokwehlukile futhi kunomthelela wendlela yokujova, ukwelashwa kokuqala kwesitshalo, kanye nengcindezi yokhunta okusetshenzisiwe. Izitshalo zomoba ezijovwe ngengcindezi ye-B. bassiana TL-leaf ngendlela yomjovo ochelelwayo wokusabalala kokhunta, kwaba nesisindo esikhulu ngaphezu kwezitshalo ezenganyelwe izinyanga ezi-3 emva kokujovwa, nesibalo esingesikhulu kangako ebudeni besitshalo uma kuqhathaniswa nezitshalo ezenganyelwe. Uma izibungu ze-E. saccharina zazivunyelwe ukunikezwa ezitshalweni izinyanga eziyi-16 emva kokujovwa nge-B. bassiana, asikho isibalo esikhulu esabonakala phakathi kwezitshalo ze-B. bassiana ekwehliseni ubude okungenamdlandla kanye nesisindo sezibungu. Okunye futhi, azikho iziqoneli ze-B. bassiana esahlukaniseka izinyanga eziyi-16 emva kokujovwa emobeni. Imiphumela yalolu cwaningo iveze ubuningi be-B. bassiana njengokhunta phakathi kohlobo lwesitshalo empilweninhlaliswano yomoba. Kugqama ukubaluleka kokwenza inhlolo yokuphilayo uma kukhethwa ingcindezi yokwengamela okuphilayo, njengoba amazinga okuvikela kanye nobungozi kwehlukile phakathi kwengcindezi ye-B. bassiana. Okunye, ucwaningo luveze ukuthi ukusebenzisa ingcindezi yokwengamela okuphilayo efanelekile ye-B. bassiana kanye nokuqonela kokuzenzela kwesikhunta komoba kuyenzeka ezinhlotsheni ezahlukene zomoba. Okunye futhi, imiphumela igcizelela ukubaluleka kokuhlonza ukuphikelela kwengcindezi yokuzenzela yomjovo we-B. bassiana njengesikhunta esitshalweni ukuqiniseka ukuthi i-B. bassiana ikhona njengesikhunta esitshalweni uma isinambuzane esiqondiwe sidla.