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CHAPTER 3 PREDISPOSI NG CAVENDISH BANANA PLANTS TO C OLD STRESS DELAYS THE DEFENCE RESPONSE AGAINS T FUSARIUM OXYSPORUM F. SP. CUBENSE ‘SUBTROPIC AL’RACE 4
ABSTRACT
Cold temperature is a major abiotic stress condition that reduces the yield of Cavendish bananas in the subtropics. It also predisposes plants to diseases such as Fusarium wilt. In this study, the hypothesis that the defence response of Cavendish bananas against Fusarium oxysporum f. sp. cubense (Foc) ‘subtropical’ race 4 (STR4), is negatively affected by low temperatures, was investigated. Greenhouse trials showed a significant increase in disease development in Cavendish bananas grown at 10oC compared to plants grown at 28oC. Numerous genes, involved in early plant response following fungal infection and cold temperature treatment, were identified using the 454 GS FLX sequencing platform. These included genes encoding pathogenesis related (PR) proteins, 1-aminocyclopropane-1-carboxylic acid oxidase, abscisic stress ripening protein, late embryogenesis abundant protein 5, metallothionein, cinnamate-4-monooxygenase, harpin-induced protein 1, lipid transfer protein, germins, peroxidase and defensins. Defence mechanisms in banana against Foc STR4 included the activation of transcripts involved in the salicylic acid, jasmonic acid and ethylene pathways. Similar transcripts were produced in Cavendish bananas exposed to 10oC and 28oC following Foc infection. However, qRT-PCR analysis showed that plant response was delayed and suppressed at the cooler temperature, thereby allowing Foc STR4 to invade the root xylem vessels and cause increased disease development. Thus, cold stress may enhance fungal infection, however disease development occurs only at 28oC, once water uptake increases. The transcriptome data obtained in this study can serve as a resource for gene expression and functional genomics studies.
INTRODUCTION
Plants are often exposed to cold stress under temperate and subtropical climatic conditions. When temperatures drop to freezing point, irreversible damage can occur on sensitive plants. Cold resistant plants, however, can withstand freezing temperatures through a process called cold acclimation (Ruelland et al., 2009). Tropical plants like banana, cucumber, mango, tomato and maize are unlikely to acclimatize to freezing temperatures and are, therefore, more sensitive to low, non-freezing temperatures (Lyons, 1973). In these plants, cold stress is a serious threat to sustainable crop production.
Low temperatures have a significant impact on bananas grown in the subtropics. Frost damage destroys the functional leaves of the plant, which reduces their photosynthetic capacity and leads to a reduction in yield. The growth of a banana plant ceases at approximately 14°C with irreversible damage occurring below freeze point (Robinson and Galán Saúco, 2010). Symptoms associated with low winter temperatures include ‘choking’, ‘choke throat’, ‘November dump’ (May flowering) as well as under-peel discolouration (Robinson and Galán Saúco, 2010). A good example of the magnitude of damage that cold stress can cause occurred in 1999, when 150 000 ha of banana plantations were destroyed in China by frost damage (Linbing et al., 2003).
Cold stress during winter does not only decrease the yield in banana, but can also predispose plants to Fusarium wilt, a disease caused by a soil-borne fungus called Fusarium oxysporum f. sp. cubense (Foc) (Viljoen, 2002). With the onset of spring, the daily temperature and transpiration rate in plants begin to rise, and disease incidence is significantly increased. Fusarium wilt (Panama disease) is considered one of the most devastating diseases of banana and has destroyed many plantations worldwide (Ploetz, 2006). Damage caused by the disease during the first half of the 20th century established it as one of the greatest epidemics in agricultural history (Ploetz and Pegg, 2000), with over $400 million (US$ 2.3 billion in 2000-value) in losses recorded in the 1950’s (Ploetz, 2005). There is no effective means to control Fusarium wilt, except for replacing susceptible banana varieties with resistant cultivars. However there is currently no resistant dessert banana variety available to replace the popular Cavendish banana, which today dominates the export and fresh fruit markets.
Cavendish bananas succumb to Fusarium wilt both in the tropics and subtropics. The variant of the fungus causing disease in the two climate zones, however, differ. In the subtropics, the disease is caused by Foc ‘subtropical’ race 4 (STR4), which belongs to vegetative compatibility group (VCG) 0120. Foc STR4 usually infects Cavendish bananas after cold predisposition, and seldom causes Fusarium wilt in Cavendish plants in tropical climates. Foc ‘tropical’ race 4 (TR4), however, does not require any predisposition by abiotic stresses for causing disease in Cavendish bananas. Foc races 1 and 2 do not cause Fusarium wilt of Cavendish bananas, neither in the subtropics nor in the tropics.
The development of Fusarium wilt can also be influenced by other abiotic stress factors also, such as hypoxia, drought, pH and salinity (Rishbeth, 1955; Simmonds, 1959; Stover, 1962). In Western Australia, the Cavendish cv. Williams showed increased disease severity under waterlogged and drought conditions after infection with Foc race 4 (Shivas et al., 1995). Low pH and high salinity favours disease development and severity in banana plants (Stover, 1962).
Despite numerous reports on the increased disease susceptibility of plants following cold stress (Line and Chen, 1995; Kim and Bockus, 2003; Bhuiyan et al., 2009), the molecular mechanisms underlying plant response is still poorly understood. This study, therefore, investigated the hypothesis that the defence response against Foc STR4 in Cavendish bananas is delayed and suppressed during cold stress. Greenhouse trials were performed to confirm the phenotypic effect of cold stress, and 454 GS FLX sequencing to identified transcripts expressed during cold stress and/or Foc infection. A subset of defence/cold stress-related genes was further studied by quantitative expression analysis.
MATERIALS AND METHODS
Fungal isolates
Foc STR4 isolates CAV 045, CAV 092 and CAV 105 (maintained at the Department of Plant Pathology, Stellenbosch University, South Africa) were cultured on half strength potato dextrose agar (PDA) and incubated for five to seven days at ± 25oC. To ensure that they were pathogenic, the isolates were first inoculated in susceptible banana plants and re-isolated from diseased rhizome material. The re-isolated cultures were then grown on half-strength PDA, and their mycelium transferred to Armstrong’s sporulation media (Booth, 1971). After five days’ growth on a shaking incubator, rotating at 120 rpm at 25oC, the spore concentration was adjusted to 1×105 spores/ml by using a hemacytometer (Laboratory & Scientific Equipment Company (Pty) Ltd. (LASEC), Randburg, South Africa).
Inoculation of banana plants
Three hundred tissue-cultured Cavendish (cv Grand Naine) banana plants were obtained from Du Roi Laboratories in Letsitele, South Africa. Grand Naine is susceptible to Foc STR4 in the subtropics, especially under stressful conditions. Plants were transplanted into a hydroponic system using 250-ml black plastic cups containing tap water, and fertilized (0.6 g/L Ca(NO3)2H2O, 0.9 g/L Agrasol, and 3 g/L Micromax) every fortnight (Nel et al., 2006). After approximately four weeks’ growth at 28oC, sufficient root growth was observed for infection.
Before inoculation, the plants were removed from the black plastic cups and their roots gently squeezed by hand to induce wounds. The plants were then replanted in polystyrene cups containing 200 ml of either a Foc STR4 spore suspension or sterile distilled water. The plants were divided into five groups and treated as follows: A. Inoculated and incubated at 28oC (infected), B. Inoculated and incubated at 10oC (coldinf), C. Incubated at 10oC for two weeks, then inoculated and transferred to 28oC (precold), D. Incubated at 10oC without inoculation (cold control) and E. Incubated at 28oC without inoculation (control). Four weeks after infection (wai), the plants incubated at 10oC (coldinf and cold) were transferred to 28oC.
Six weeks after inoculation, the rhizomes of the banana plants were dissected horizontally and the degree of discolouration determined according to the INIBAP rating scale (Carlier et al., 2002). Disease severity was calculated from ten plants per treatment as: DSI% = Σ (number of scale x number of plants in that scale)/Σ (number of treated plants) (Sherwood and Hagedorn, 1958). All the data were analyzed by JMP® (SAS Institute, Cary, North Carolina) using analysis of variance (ANOVA) test with significant difference values at p<0.05 using the Student t-test. The plants used for the quantitative reverse transcriptase PCR (qRT-PCR) were stripped of most of their roots and placed back into the cups. Eight to ten weeks later the rhizome was sliced open and used to determine disease severity as described above.
RNA extraction and cDNA generation
For transcriptome analysis, roots were harvested at 3 and 12 hours post infection (hpi), while for qRT-PCR, roots were harvested at 0, 3, 12, 24, 48 hpi and five days post infection (dpi). The roots were then rapidly frozen in liquid nitrogen and stored at -80oC. RNA was extracted from the roots of six plants each that were collected 3 and 12 hpi. The RNA of the two collection points was then combined for each of the treatments (infected, coldinf and precold) to obtain a representative sample of early plant response after infection. For quantitative gene expression studies, RNA (60 µg) from two plants was combined of each time point and regarded as a biological repeat.
RNA was extracted from banana roots with a CTAB extraction buffer and LiCl precipitation (Chang et al., 1993). For transcriptome analysis, 360 µg RNA was treated with DNaseI (Fermentas, Life Sciences, Hanover, USA) and purified with a RNeasy mini kit (Qiagen, Valencia, California, USA). The quantity of RNA was determined with a Nanodrop ND-100 Spectrophotometer (Nanodrop Technologies, Inc., Montchanin, USA) and quality was assessed by gel electrophoresis under non-denaturing conditions on 2% agarose gel. mRNA was isolated from total RNA using the oligotex mRNA mini kit (Qiagen) according to the manufacturer’s instructions. From the mRNA, double stranded cDNA was synthesized with cDNA Synthesis System (Roche Diagnostics, Mannheim, Germany). For quantitative gene expression studies, 60 µg RNA was treated with DNaseI and purified with an RNeasy mini kit. Single-stranded cDNA was synthesized using the Transcriptor first strand cDNA synthesis kit (Roche Diagnostics). In both cases, DNA contamination was verified by PCR using intron flanking actin primers for plant cDNA (Van den Berg et al., 2007).
DECLARATION
ACKNOWLEDGEMENTS
PREFACE
ABBREVIATIONS AND SYMBOLS
LIST OF TABLES.
LIST OF FIGURES
CHAPTER 1: THE EFFECT OF COLD STRESS ON PLANT DISEASE DEVELOPMENT, WITH SPECIAL REFERENCE TO FUSARIUM WILT OF BANANA
INTRODUCTION
COLD STRESS IN PLANTS
ELEVATED LEVELS OF DISEASE INCIDENCE AND DEVELOPMENT DUE TO TEMPERATURE
FACTORS THAT GIVE RISE TO INCREASED DISEASE INCIDENCE WITH A CHANGE IN TEMPERATURE
BANANA, A TROPICAL PLANT
FACTORS INFLUENCING THE DEVELOPMENT OF FUSARIUM WILT OF BANANA
APPROACHES TO IMPROVE COLD TOLERANCE IN PLANTS
CONCLUSION
REFERENCES
TABLES AND FIGURES
CHAPTER 2: PATHOGENICITY ASSOCIATED GENES IN FUSARIUM OXYSPORUM F. SP. CUBENSE RACE 4
ABSTRACT .
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
TABLES AND FIGURES
CHAPTER 3: PREDISPOSING CAVENDISH BANANA PLANTS TO COLD STRESS DELAYS THE DEFENCE RESPONSE AGAINST FUSARIUM OXYSPORUM F. SP. CUBENSE ‘SUBTROPICAL’ RACE 4
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS.
RESULTS
DISCUSSION
CONCLUSIONS
TABLES AND FIGURES
CONCLUSIONS
REFERENCES
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