Jaindra Nath Tripathi

A rapid technique for screening banana cultivars for resistance to Xanthomonas wilt

The banana Xanthomonas wilt disease (BXW) has threatened the livelihood of millions of farmers in East Africa. Use of resistant varieties is the most cost-effective method of managing this bacterial disease. A reliable and rapid screening method is needed to select resistant banana varieties. An in vitro screening method was developed for early evaluation of Xanthomonas wilt resistance using small tissue culture-grown plantlets. Eight cultivars of banana were screened with sixteen isolates of Xanthomonas campestris pv. musacearum using this method. There were significant differences (P < 0.0001) in susceptibility among the various banana cultivars tested, whereas no significant difference (P = 0.92) in pathogenicity was observed between the pathogen isolates. The cv. Pisang Awak (Kayinja) was found to be highly susceptible and Musa balbisiana resistant. Nakitembe was found to be moderately resistant while cvs Mpologoma, Mbwazirume, Sukali Ndiizi, FHIA-17 and FHIA-25 were susceptible. The susceptibility of these cultivars was further tested in vivo by artificial inoculation of potted plants with similar results. This study shows that an in vitro screening test can serve as a convenient, cheap and rapid screening technique to discriminate BXW-resistant from BXW-susceptible banana cultivars.

Generation of transgenic plantain (Musa spp.) with resistance to plant pathogenic nematodes

Plant parasitic nematodes impose a severe constraint on plantain and banana productivity; however, the sterile nature of many cultivars precludes conventional breeding for resistance. Transgenic plantain cv. Gonja manjaya (Musa AAB) plants, expressing a maize cystatin that inhibits nematode digestive cysteine proteinases and a synthetic peptide that disrupts nematode chemoreception, were assessed for their ability to resist nematode infection. Lines were generated that expressed each gene singly or both together in a stacked defence. Nematode challenge with a single species or a mixed population identified 10 lines with significant resistance. The best level of resistance achieved against the major pest species Radopholus similis was 84% ± 8% for the cystatin, 66% ± 14% for the peptide and 70% ± 6% for the dual defence. In the mixed population, trial resistance was also demonstrated to Helicotylenchus multicinctus. A fluorescently labelled form of the chemodisruptive peptide underwent retrograde transport along certain sensory dendrites of R. similis as required to disrupt chemoreception. The peptide was degraded after 30 min in simulated intestinal fluid or boiling water and after 1 h in nonsterile soil. In silico sequence analysis suggests that the peptide is not a mammalian antigen. This work establishes the mode of action of a novel nematode defence, develops the evidence for its safe and effective deployment against multiple nematode species and identifies transgenic plantain lines with a high level of resistance for a proposed field trial.

Agrobacterium tumefaciens-Mediated Transformation of Pseudocercospora fijiensis to Determine the Role of PfHog1 in Osmotic Stress Regulation and Virulence Modulation

Black Sigatoka disease, caused by Pseudocercospora fijiensis is a serious constraint to banana production worldwide. The disease continues to spread in new ecological niches and there is an urgent need to develop strategies for its control. The high osmolarity glycerol (HOG) pathway in Saccharomyces cerevisiae is well known to respond to changes in external osmolarity. HOG pathway activation leads to phosphorylation, activation and nuclear transduction of the HOG1 mitogen-activated protein kinases (MAPKs). The activated HOG1 triggers several responses to osmotic stress, including up or down regulation of different genes, regulation of protein translation, adjustments to cell cycle progression and synthesis of osmolyte glycerol. This study investigated the role of the MAPK-encoding PfHog1 gene on osmotic stress adaptation and virulence of P. fijiensis. RNA interference-mediated gene silencing of PfHog1 significantly suppressed growth of P. fijiensis on potato dextrose agar media supplemented with 1 M NaCl, indicating that PfHog1 regulates osmotic stress. In addition, virulence of the PfHog1-silenced mutants of P. fijiensis on banana was significantly reduced, as observed from the low rates of necrosis and disease development on the infected leaves. Staining with lacto phenol cotton blue further confirmed the impaired mycelial growth of the PfHog1 in the infected leaf tissues, which was further confirmed with quantification of the fungal biomass using absolute- quantitative PCR. Collectively, these findings demonstrate that PfHog1 plays a critical role in osmotic stress regulation and virulence of P. fijiensis on its host banana. Thus, PfHog1 could be an interesting target for the control of black Sigatoka disease in banana.

Genetically engineered bananas resistant to Xanthomonas wilt disease and nematodes

Abstract Banana is an important staple food crop feeding more than 100 million Africans, but is subject to severe productivity constraints due to a range of pests and diseases. Banana Xanthomonas wilt caused by Xanthomonas campestris pv. musacearum is capable of entirely destroying a plantation while nematodes can cause losses up to 50% and increase susceptibility to other pests and diseases. Development of improved varieties of banana is fundamental in order to tackle these challenges. However, the sterile nature of the crop and the lack of resistance in Musa germplasm make improvement by traditional breeding techniques either impossible or extremely slow. Recent developments using genetic engineering have begun to address these problems. Transgenic banana expressing sweet pepper Hrap and Pflp genes have demonstrated complete resistance against X. campestris pv. musacearum in the field. Transgenic plantains expressing a cysteine proteinase inhibitors and/or synthetic peptide showed enhanced resistance to a mixed species population of nematodes in the field. Here, we review the genetic engineering technologies which have potential to improve agriculture and food security in Africa.

Transgenic Technologies for Bacterial Wilt Resistance

Banana production is severely affected by bacterial diseases jeopardizing the food security of millions of inhabitants in countries where farmers depend upon banana as staple food. Bacterial diseases like Xanthomonas wilt, Moko, blood, and Bugtok are the most important diseases threatening banana cultivation in several tropical and subtropical countries. Genetic improvement of banana through classical breeding is difficult due to the lack of resistant germplasm, sterile nature, and long generation time. Transgenic technology can complement classical breeding for developing bacterial disease-resistant varieties. Some success has been achieved for developing host plant resistance in order to control banana Xanthomonas wilt (BXW) disease. Currently, the transgenic bananas expressing either sweet pepper Pflp or Hrap gene are under evaluation for resistance to Xanthomonas wilt disease in field trials in Uganda. Management of bacterial diseases through cultural practices like removal of male buds and use of pathogen-free seed material and disinfected cutting tools can contain outbreak of diseases although these are not absolute solutions for control of bacterial diseases. In this chapter, we have discussed various management practices as well as existing transgenic technologies to control bacterial diseases of banana.

High-Frequency Shoot Regeneration of Various Cultivars of Banana (Musa sp.)

ABSTRACT An efficient, simple, and rapid regeneration system was established for bananas (Musa sp.) using sections of corm containing intercalary meristematic tissues as explants. Six different cultivars of banana (‘Mpologoma’, ‘Nakitembe’, ‘Mbwazirume’, ‘Pisang awak’, ‘Sukali ndiizi’, and ‘FHIA-17’) with diverse genetic constitution and ploidy levels were regenerated on Murashige and Skoog (MS) medium supplemented with 5 mgl−1 of 6-benzylaminopurine. All the cultivars showed relatively high regeneration efficiency. About 93–97% of the explants regenerated, producing, on average, 12–13 shoots from whole section and 16–19 shoots in total from quarter pieces of each section. Regeneration from intercalary meristematic tissues offers a simple, efficient method for transformation of a broad range of banana cultivars, including East African Highland bananas.

Silencing of the Mitogen-Activated Protein Kinases (MAPK) Fus3 and Slt2 in Pseudocercospora fijiensis Reduces Growth and Virulence on Host Plants

Pseudocercospora fijiensis, causal agent of the black Sigatoka disease (BSD) of Musa spp., has spread globally since its discovery in Fiji 1963 to all the banana and plantain growing areas across the globe. It is becoming the most damaging and economically important disease of this crop. The identification and characterization of genes that regulate infection processes and pathogenicity in P. fijiensis will provide important knowledge for the development of disease-resistant cultivars. In many fungal plant pathogens, the Fus3 and Slt2 are reported to be essential for pathogenicity. Fus3 regulates filamentous-invasion pathways including the formation of infection structures, sporulation, virulence, and invasive and filamentous growth, whereas Slt2 is involved in the cell-wall integrity pathway, virulence, invasive growth, and colonization in host tissues. Here, we used RNAi-mediated gene silencing to investigate the role of the Slt2 and Fus3 homologs in P. fijiensis in pathogen invasiveness, growth and pathogenicity. The PfSlt2 and PfFus3 silenced P. fijiensis transformants showed significantly lower gene expression and reduced virulence, invasive growth, and lower biomass in infected leaf tissues of East African Highland Banana (EAHB). This study suggests that Slt2 and Fus3 MAPK signaling pathways play important roles in plant infection and pathogenic growth of fungal pathogens. The silencing of these vital fungal genes through host-induced gene silencing (HIG) could be an alternative strategy for developing transgenic banana and plantain resistant to BSD.