Fusarium Head Bright in Wheat with Aphids Herbivory - Lab Report Example

Fusarium Head Bright in Wheat with Aphids Herbivory. Student Name: Course Lecturer University Town Date Abstract Plant diseases are a major threat to food security in the world. One of the fungal disease that attacks cereal crops and causes a danger to food quality and safety is Fusarium. Fusarium Head Blight (FHB) reduces cereal crops yield and hence there is a need to understand how to control the infections and factors that increases the susceptibility of wheat to Fusarium Head Blight. The current study examined how aphid infestation affect the disease severity of two varieties of wheat. The study found that aphid increases the susceptibility of wheat to Fusarium infection. In particular, the Fusarium infection is severe when aphids are applied on the ear than on the leaves. Furthermore, the Gallant variety is more susceptible to Fusarium infection than the Sumai 3 variety. Introduction Plant diseases are a major obstacle to food security in the world. Diseases such as Fusarium head blight affect not only food quality and safety but also directly impact on human and animal health (Chakraborty & Newton 2011). Fusarium head blight is a cereal crop disease that is capable of reducing crop yield due to the mycotoxin they produce (Walter et al. 2010). There are two species of Fusarium; Fusarium culmorum and Fusarium graminearum. Fusarium culmorum causes seedling bright, foot rot and Fusarium head bright (FHB) in wheat (Wagacha & Muthomi 2007). Given that the global food production need to increase by 50% to meet the world demand at 2050, there is a need to understand how the FHB can be controlled and factors that amplify the infections (Chakraborty & Newton 2011). There is evidence that grains aphids, Sitobion avenae increases the susceptibility of wheat to Fusarium langsethiae and aphids infestation is associated with high levels of T-2 and HT-2 mycotoxins in wheat products (Drakulic et al. 2016). This paper offer more evidence on how aphid’s infestation affects Fusarium infection in wheat. Materials and Methods Plant material: Seeds of winter wheat (Triticum aestivum) cv. ‘Gallant’ and spring wheat cv. ‘Sumai 3’ were used. ‘Gallant’ seeds were treated with Redigo Deter (TM) and sown in compost (Levington’s F2+S) individually into module trays (2 cm x 2 cm x 5 cm, l x w x d, per module) and allowed to germinate in a glasshouse at minimum 15, maximum 18°C daytime temperature and minimum 12, maximum 15°C night time temperature for two weeks under a 12h photoperiod. Seedlings were transferred to a polytunnel and kept at 4°C for 6-8 weeks for vernalisation. ‘Sumai 3’ seeds were sown at the same time as ‘Gallant’ seedlings were potted on, with one seed or seedling being planted directly into compost (John Innes Type 2) in individual 5 L pots, and fed by automatic irrigation in the glasshouse conditions described above until ear emergence. F. graminearum inoculum: Isolates of F. graminearum were obtained from single spores cultures from the University of Nottingham isolate collection, (#212, 214 and 216). Isolates were grown on PDA for 7-10 days then on synthetic nutrient deficient agar (SNA) for a further 14 days to initiate spore production. SNA cultures were then washed with 1 ml sterile distilled water (SDW), agitated with a sterile L-shaped spreader and harvested by pipette. The concentration of harvested spores was calculated using a haemocytometer (Neubauer) and adjusted to 250,000 spores. ml-1 and each isolate combined in equal proportions to make a composite inoculum. Aphid rearing: English grain aphids, Sitobion avenae, originally from colonies maintained at Rothamsted Research (Harpenden, UK) were used to initiate colonies within rearing cages at the University of Nottingham. Rearing cages were maintained in the glasshouse conditions described above, and aphids were reared on several winter wheat cv. ‘Gallant’ seedlings grown from unvernalised surface sterilised seeds grown in 5l pots and caged within a metal framed net cage that attached directly to the pot (Insectopia (TM), Watkins & Doncaster, Bolton, UK). Treatment structure: The experiment was performed as a factorial design in randomised blocks. Two different varieties of wheat were used: ‘Gallant’ (susceptible to FHB) and ‘Sumai 3’ (Type II resistant to FHB i.e. resists spread throughout ear). The two varieties were either inoculated or mock inoculated. Inoculation was performed on two tillers per plant by point inoculation at the 10th spikelet, as counted from the bottom up, with 25 µl of either spore suspension or SDW for mock inoculated controls. Following inoculation or mock inoculation, ears were bagged in perforated plastic bags for 48h. Inoculation was applied at mid-anthesis (GS65) (Zadoks et al., 1974). Three aphid treatments were used: (1) Treatment ‘N’, aphid-free controls; (2) Treatment ‘L’, aphids applied to the flag leaf for 7d prior to inoculation (systemic treatment), (3) Treatment ‘E’, aphids applied to ears for a period of 7d before inoculum was applied (local treatment). Adult aphids (n = 10) were added to leaves using a brush and trapped on the adaxial side of the leaf using leaf clips (inner radius = 2 cm). Aphids applied to ears (n = 10) were of similar age and trapped on the ears using netting. Seven days after aphid application, leaf clips and nets were removed and all plants were sprayed using 500 g/kg (50% w/w) pirimicarb (Aphox, Syngenta). The full treatment structure was replicated 6 times and is detailed in full in Table 1. Visual disease assessment was performed on 5 intervals: 5 days after inoculation (DAI), 10, 16, 21 and 28 DAI. The number of visible symptoms (either dark lesions or bleached spikelets) that were observed on ears was recorded as the number of affected spikelets, which was divided by the total number of spikelets per ear to calculate the disease severity (%) and from this data the area under the disease progress curve (AUDPC) was also calculated. Estimation of fungal biomass by QPCR: Ears were harvested at maturity, freeze dried whole and milled without threshing using a centrifugal mill (ZM 200; Retsch GmbH, Germany) with a sieve size of 1 mm. DNA was extracted from flour samples (0.5 g) which were firstly added to CTAB buffer (3.75 ml), heated in a water bath for 2 h, chilled on ice, then added to potassium acetate (1.25 ml). Tubes were mixed by inversion, centrifuged at 2000 xg for 15 mins and 1.2 ml supernatant removed and added to 600 µl chloroform (100%). The rest of the method proceeded as previously described (Edwards et al., 2001), and the final DNA pellets were resuspended using 200 µl TE buffer in a hot block at 65°C for 2 h. Quantification of DNA concentration was performed by measuring the absorbance of light at wavelength 260, 280 and 328 nm using a Cary 50 Probe UV-visible spectrophotometer (Varian, CA, USA). DNA stocks were diluted to 20 ng. ml-1 and used in QPCR assays with F. graminearum specific primers (Fg16NF 5’ - ACA GAT GAC AAG ATT CAG GCA CA -3’; Fg16NR 5’- TTC TTT GAC ATC TGT TCA ACC CA – 3’) using a thermocycling program of: 90 s at 95°C; 35 cycles of 30 s at 94°C, 45 s at 64°C, 45 s at 72°C; and 5 min at 72°C which produced a 280 bp product (Nicholson et al., 1998). Quantification of pathogen DNA concentration as a proportion of DNA in the flour extracts was performed by quantitative real-time PCR (QPCR) using a total volume of 13 µl comprising 2.5 µl sample DNA, SYBR green 2X mastermix (Bio-Rad, USA) and 250 nM F. graminearum primers. DNA extracted from a pure culture of isolate #216 from the University of Nottingham isolate collection was used to prepare a standard curve of ten-fold dilutions ranging from 1 to 1 x 10-6 ng. µl-1, and linear regression was used to calculate the starting quantity of target DNA in each sample from the standard curve. The limit of detection for the assay was 0.0001 pg.ng-1. RESULTS Disease severity at day 4: ANOVA analysis of the disease severity for wheat that was inoculated with Fusarium shows that there was significant difference in the means of the aphid infested wheat and also in the different varieties of the wheat. The means of disease severity for the aphid infested wheat were significantly different with a p-value of 0.017. For the two varieties of wheat, the means of the disease severity were significantly different with a p-value of 0.001. For the interaction of the wheat varieties and the aphid infestation, the means were not statistically different from each other with a p-value of 0.69. Disease severity at day 10: ANOVA analysis of the disease severity for wheat that was inoculated with Fusarium shows that there was significant difference in the means of the aphid infested wheat. The means of disease severity for the aphid infested wheat was significantly different with a p-value of 0.001. For the two varieties of wheat, the means of the disease severity were significantly different with a p-value of 0.042. For the interaction of the wheat varieties and the aphid infestation, the means were not statistically different from each other with a p-value of 0.616. Disease severity at day 16: Analysis of the disease severity for wheat that was inoculated with Fusarium shows that there was significant difference in the means of the aphid infested wheat. The means of disease severity for the aphid infested wheat were significantly different with a p-value of 0.001. For the two varieties of wheat, the means of the disease severity were significantly different with a p-value of 0.001. However, the means of the interaction of the wheat varieties and the aphid infestation, were not statistically different from each other with a p-value of 0.079. Disease severity at day 21: Analysis of the disease severity for wheat that was inoculated with Fusarium shows that there was significant difference in the means of the aphid infested wheat and the two varieties. The means of disease severity for the aphid infested wheat were significantly different with a p-value of 0.001. For the two varieties of wheat, the means of the disease severity were significantly different with a p-value of 0.001. However, the means of the interaction of the wheat varieties and the aphid infestation, were not statistically different from each other with a p-value of 0.617. Disease severity at day 28: Analysis of the disease severity for wheat that was inoculated with Fusarium shows that there was significant difference in the means of the aphid infested wheat. The means of disease severity for the aphid infested wheat were significantly different with a p-value of 0.001. For the two varieties of wheat, the means of the disease severity were significantly different with a p-value of 0.001. However, the means of the interaction of the wheat varieties and the aphid infestation, were not statistically different from each other with a p-value of 0.623. Area under disease progress curve: Analysis of the AUDPC for wheat that was inoculated with Fusarium shows that there was significant difference in the means of the AUDPC of aphid infested wheat. The means of AUDPC of the aphid infested wheat were significantly different with a p-value of 0.001. For the two varieties of wheat, the means of the AUDPC were significantly different with a p-value of 0.001. However, the means of the interaction of the wheat varieties and the aphid infestation, were not statistically different from each other with a p-value of 0.487. Pathogen DNA: To study the concentration of the pathogen DNA in the host final output, the amount of the DNA is log transformed. Analysis of the concentration of DNA of the pathogen for wheat that was inoculated with Fusarium shows that there was significant difference in the means of the aphid infested wheat. The means of DNA concentration for the aphid infested wheat were significantly different with a p-value of 0.001. For the two varieties of wheat, the means of the DNA concentration were significantly different with a p-value of 0.001. However, the means of the interaction of the wheat varieties and the aphid infestation, were not statistically different from each other with a p-value of 0.255. However, when the mock inoculation was included the difference in the means disappeared. Discussion The results shows that aphid infestation significantly increases the level of Fusarium infection in wheat. Furthermore, the Fusarium infection is more severe if the aphids are applied on the ear than when applied on the leaves. At day 4, the disease severity of wheat with aphids on the ear (8.29) is almost double of those with no aphid infestation (4.43). The aphid infestation increases the susceptibility of wheat to Fusarium both at early stages of infections and at the late stages (Drakulic et al. 2016). Furthermore, Gallant variety of wheat is more susceptible to Fusarium infection than Sumai 3 variety. The disease severity at day 4 for Gallant (8.8) is almost twice that of the Sumai 3 (4.84). The disease severity for aphid infested and the two variety of the wheat increases with the number of days. For the area under disease progress curve, the disease progress was highest for the wheat with aphids on the ear and the least for the wheat with no aphids. This is in line with the above observation of the disease severity increasing with the number of days. Moreover, the Gallant variety has a higher area under the curve than the Sumai 3. The results indicates that aphid helps in the Fusarium infection. The results for the DNA concentration shows that wheat with aphid ear infestation has a higher amount of the DNA of Fusarium compared to wheat with no aphids. For pathogen DNA concentrations, aphid-free inoculated wheat had a mean of -0.212 compared to a mean of 0.376 in inoculated wheat to which aphids were added to the ear. The concentration of DNA of the pathogen also differed with the wheat variety. Gallant variety had a higher mean concentration of 0.418 compared to Sumai 3 with -0.179 References Chakraborty, S. & Newton, A.C., 2011. Climate change , plant diseases and food security : an overview. Plant Pathology, 60, pp.2–14. Drakulic, J. et al., 2016. Aphid Infestation Increases Fusarium langsethiae and T-2 and HT-2 Mycotoxins in Wheat. Applied and Environmental Microbiology, 82(22), pp.6548–6556. Wagacha, J.M. & Muthomi, J.W., 2007. Fusarium culmorum : Infection process , mechanisms of mycotoxin production and their role in pathogenesis in wheat. Crop Protection, 26, pp.877–885. Walter, S., Nicholson, P. & Doohan, F.M., 2010. Action and reaction of host and pathogen during Fusarium head blight disease. New Phytologist, 185, pp.54–66. Figures and Tables Analysis of variance Aphid*Variety*Inoculation (nothing excluded) Variate: Log10DNA Source of variation d.f. s.s. m.s. v.r. F pr. Block stratum 5 1.957 0.3914 0.66 Block.*Units* stratum Aphid 2 7.831 3.9155 6.63 0.002 Variety 1 0.3839 0.3839 0.65 0.422 Inoculation 1 173.9177 173.9177 294.49 Read More