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Flooding caused or exacerbated by climate change has threatened plant growth and food production worldwide. The lack of knowledge on how crops respond and adapt to flooding stress imposes a major barrier to enhancing their productivity. Hence, understanding the flooding-responsive mechanisms of crops is indispensable for developing new flooding-tolerant varieties. Here, we examined the banana (Musa acuminata cv. Berangan) responses to soil waterlogging for 1, 3, 5, 7, 14, and 24 days. After waterlogging stress, banana root samples were analyzed for their molecular and biochemical changes. We found that waterlogging treatment induced the formation of adventitious roots and aerenchyma with conspicuous gas spaces. In addition, the antioxidant activities, hydrogen peroxide, and malondialdehyde contents of the waterlogged bananas increased in response to waterlogging stress. To assess the initial response of bananas toward waterlogging stress, we analyzed the transcriptome changes of banana roots. A total of 3508 unigenes were differentially expressed under 1-day waterlogging conditions. These unigenes comprise abiotic stress-related transcription factors, such as ethylene response factors, basic helix-loop-helix, myeloblastosis, plant signal transduction, and carbohydrate metabolisms. The findings of the study provide insight into the complex molecular events of bananas in response to waterlogging stress, which could later help develop waterlogging resilient crops for the future climate.

Musa acuminata cv. Berangan (AAA) is a type of banana locally grown in Malaysia. These bananas as well as Musa acuminata cv. Cavendish (AAA) are also facing a major threat from a typical soil borne fungus identified as Fusarium oxysporum f. sp. cubense race 4 (FocR4). Its characteristics as a complex pathogen manifesting as subtypes or races are the main reasons its infections are difficult to control. Genome sequence availability of the double haploid Musa acuminata originating from Pahang has become very useful to analyse RNA-seq reads and to identify the transcriptome profile of the host response between different groups was accomplished using RNA-Seq technology based on the Illumina HiSeqTm 2000 platform. Three sets of libraries derived from infected and mock infected plants (experimental groups) between different time points (0, 48, 96 h) shows over forty million reads were generated, each corresponding to coverage of >4,000,000,000 to <8,000,000,000 bases. About 0.10-66% reads were mapped to Musa acuminata DH Pahang genome sequence. This study provides the statistical analysis of the sequence reads. Based on this information, further analysis on gene expression patterns influenced by Foc race infection within the tested groups and time points will help in the understanding of the host pathogenic responses. In future discovery on many new genes for diagnosis of plant infection could be achieved through excessive transcriptomic data.

Respiratory burst oxidase homologs (RBOH) is the key enzyme responsible for the production of reactive oxygen species which act as important signal during plant responses to abiotic and biotic stresses. However, RBOH homologs have not been characterized in banana. In this study, we have identified twelve Rboh genes distributed on eight chromosomes of Musa acuminata subsp. malaccensis (DH Pahang) through a genome-wide analysis. MaRbohs exist as sibling paralogs with variable exon-intron structures and highly conserved functional domain. Phylogenetic analysis clustered MaRBOH into four distinct subgroups (I, II, III and IV). The expression of MaRbohs following 24 hours of inoculation with Fusarium oxysporium f. sp. cubense Tropical Race 4 revealed diverse patterns in root tissues. MaRbohH1 and MaRbohA2 were strongly upregulated and downregulated, respectively in response to the root-dip inoculation. This is the first report on genome-wide characterization of Rboh genes from banana and their expression in response to the fungal pathogen TR4. This research provides a basis for exploration of the role of NADPH oxidase in banana defence against the root pathogen.

Fusarium oxysporum is one of the most adaptive and pathogenic fungus that causes wilting in banana. It is regarded as one of the economically destructive fungus and control of this disease is always ineffective once the disease becomes established in banana plantations. Various researches have been carried out, but sustainable solution is yet to achieve. In this study, we identified the infection phenotypically through LSI and RDI which can be only done after 5th-week of post inoculation. With qPCR application, we identified the infection level much faster within 2 to 96 hrs. Disease severity index obtained from LSI and RDI value showed that Berangan is highly susceptible towards C1 HIR, 9888 and 2296. Meanwhile, qPCR analysis besides showing infection pattern as early as 2 hrs in Berangan was infected with C1 HIR, 9888 and 2296. We also found that additional information such as specific genes were expressed in the Berangan infected with C1 HIR, 9888 or 2296. This information can be used to identify the fusarium strain that is actually infecting Berangan. This could help planters to identify early infection of fusarium as well as strain recognition.

To date, there is no standardized Fusarium bioassay protocol established owing partly to the wide variety of Fusarium oxysporum f. sp. cubense (Foc) isolates and banana cultivars present. Thus, validation of the infection parameters is deemed essential prior to each bioassay experiment. In the current study, a simple standardized workflow was developed based on available assays for testing Fusarium wilt disease response in Musa acuminata using M. acuminata cv. ‘Berangan’ of tissue-culture origin as a model. The phenotypic assays were able to detect external disease symptoms less than one week post-inoculation, while the molecular approach using RT-qPCR identified differential expression of catalase (CAT), pathogenesis-related 10 (PR10), phenylalanine ammonia-lyase (PAL) and xylanase (XYL) genes as early as day 0. The transcript levels of PR10 and XYL fluctuated over 4 days of Foc Race 4 (FocR4 C1 HIR isolate) infection while the expression of CAT steadily increased over time. In contrast, PAL was highly upregulated at 2 days post-inoculation. These signature changes suggest that all genes tested might be involved in the early defense response of ‘Berangan’ plants against FocR4 infection. ‘Berangan’ cultivar was found to be highly susceptible to Foc Race 4 (C1 HIR isolate) with leaf symptoms index (LSI) and rhizome discoloration index (RDI) scores of 4.257 and 5.971, respectively. The procedure elaborated in this study can be used as a reference Foc bioassay for reproducible and comparable results possibly across cultivars and test isolates due to its simple steps aided by integration of phenotypic and molecular approach.

The expression profile of pathogenesis related genes are signatures of an infection response in plant cells. Pathogenic infections can increase or reduce gene expression in a plant system in a relatively specific pattern. These expression patterns can be used as standards in pathogenicity studies and, where phenotypic expression is normally used to gauge a plants response to infection, it could additionally present a more rapid and early screening reference tool. Three genes: catalase (CAT), pathogen related protein (PR10), and phenylalanine ammonia (PAL) all implicitly implicated in the plant disease response pathway were targeted for analysis during the infection of Fusarium oxysporum f. sp. cubense tropical race 4 (FOCR4) in banana Musa acuminata cv. Berangan seedlings after a standard challenge under growth room conditions. Distinct patterns of gene expression were observed at three infection time points by real time expression analysis. There was a sequential 10-fold reduction in expression for the PR gene while, the PAL and CAT genes were both upregulated. These results present a set of reference genes that could be used for screening of a plant’s response to Fusarium before the onset of symptoms.

PR-10 is a member of pathogenesis-related (PR) genes elicited by the plant’s defense mechanism during pathogen attack. Elevated expression of PR-10 upon different pathogen invasions has been observed in many plant species suggesting its role as an anti-bacterial, anti-viral and anti-fungal gene. However, the effect of PR-10 in mitigating the infection of Fusarium oxysporum f. sp. cubense (Foc), the causal agent of Fusarium wilt in banana has not been reported. In this study, the coding sequences of PR-10 gene isolated from Foc resistant Musa acuminata ssp. malaccensis (MaPR-10) were integrated into a local Foc susceptible commercial banana cultivar, Berangan via co-cultivation of embryogenic cell suspension and Agrobacterium tumefaciens. Out of 17 putative transgenic lines established, 11 of them positively harbored MaPR-10. Among these, Line-19 plantlets showed the most rapid in-vitro propagation and successfully over- expressed the transgene. Following a nursery challenge experiment with a virulent Foc race 4 (CI HIR) isolate, about 30% of Line-19 plants showed a one-week delay in disease progression when compared to the untransformed controls. From the final evaluation performed in the 5th week-post-inoculation, the leaf symptoms index (LSI) and rhizome discoloration index (RDI) of Line-19 was 3.4 and 6.1, respectively, indicating the disease had progressed. The findings of this study enrich the current existing knowledge on the roles of PR-10 in combating fungal disease in plants.

Pisang Berangan, is the most popular dessert banana cultivar in Malaysia. This AA type is grouped within highly nutritious type of fruits. Therefore, its production to cater for human needs is crucial. However, fusarium wilt disease is threatening world banana production. Availability of banana transcriptome will be highly useful for improving banana gene annotation and for biological research. The knowledge of global gene expression patterns that are influenced by the Fusarium races, the varieties of infected banana cultivar and the environmental conditions will help to provide better understanding on the host responses towards the infection. RNA samples from laboratory infected Berangan were pooled for transcriptomics using the Illumina Technology. Analysis of the banana transcriptome led to the identification of overexpressed genes. Differential gene expression (DGE) profiling analysis reveals that the infection by Fusarium caused changes in the global gene expression profiles in the banana roots 2 hrs post infection. The infection led to the induction of many well known defense related genes. This transcriptome profiling analysis indicates that early detection is possible and through this any infected plant can be identified and further essential precaution can be carried out.