Phenotypic variables measured Helena L. Brochero embrochero@unal.edul.co Faculty of Agricultural Sciences Universidad Nacional de Colombia Bogotá, Colombia Bio-Protection Research Centre Lincoln University Lincoln, New Zealand Michael Rostás, Travis Glare, Maya Raad, Aimee McKinnon Bio-Protection Research Centre, PO Box 85084, Lincoln University, Lincoln 7647, New Zealand Endophytic lifestyle of an insect pathogen causes isolate-specific differences in the development of the host plant but not in its herbivores Background Beauveria bassiana can act as entomopathogenic fungus killing insect populations or as true endophyte by colonizing plant tissues contributing to induced plant defence responses. We assessed the effects of two B. bassiana isolates from New Zealand on the development of Arabidopsis thaliana Col-1. We evaluated the performance of a leaf-chewing (Plutella xyllostella) and a phloem-sucking herbivore (Myzus persicae) when fed with endophyte-infected plants. Methodology Fifteen -day old Arabidopsis thaliana Col-1 plants were inoculated with 108 spores/mL solutions of B. bassiana strains BG11 and FRh2 by root dip inoculation. Control plants were inoculated with 0.05% Tween 80. Four and twelve weeks after inoculation, 11 different phenotypical variables were measured per plant (n=20 per group each time) (Boyes et al., 2001). Fungus colonization was assessed through plating on a selective medium with antibiotics and qPCR using B. bassiana-specific markers (Castrillo et al., 2003) Bioassays were carried out to test the effect of plant-endophyte association on the growth of Plutella xylostella caterpillars and on the fecundity of the aphid Myzus persicae. 1 2 Figure 1: Discriminant analysis representing variables between phenotypical traits in groups of Arabidopsis thaliana plants inoculated with Bauveria bassiana BG11 and FRh2 strains A Figure 2: Effect of endophytic B. bassiana on herbivores: (A-C) Endophytic B. bassiana effect on P. xylostella growth post-infestation with third instar caterpillars. Error bars represent the standard error of the mean (N=15-20). Caterpillars of the same age were used within one experiment. (D) Endophytic B. bassiana effect on M. persicae population after 10 days post infestation. Error bars represent the standard error of the mean (N=12-14). No significant differences between treatments (P < 0.05). n.s. = not significant. n.s. (A) (B) (C) (D) 2 1 BG11-inoculated plants showed delayed plant development affecting all growth stages. However, BG11-inoculated plants had greater plant biomass. Likewise, seeds from BG11-inoculated plants had slower germination rate and less uniform germination. FRh2-inoculated plants were not significantly different to control plants in terms of phenotypic traits and developmental time. Likewise, controls and FRh2 –inoculated A. thaliana plants have similar rates of seed production and germination. Endophyte presence did not have any antagonistic effects on the growth of P. xylostella and the fecundity of M. persicae (Figure 2). However the re-isolated fungus caused 100% mortality when applied topically on caterpillars. Results Group   Phenotypic variables measured stem legth2 rosette radius no leaves2 shoot f.w root legth root f.w root dry.w ratio r.s.f.w stem daysb no leavesb stem lengthb CONTROL Mean 17.704 105.750 52.5 4.300 95.895 2.832 0.332 0.644 28.5 12.95 28.35 Median 16.65 104.735 54.5 4.337 93.915 2.738 0.333 0.606 30.00 13.00 32.545 Standard deviation 10.627 18.076 8.532 1.0919 18.297 1.492 0.163 0.281 4.335 1.317 23.915 Sample variance 112.933 326.757 72.789 1.192 334.766 2.226 0.026 0.079 18.790 1.734 571.936 Kurtosis -0.919 -0.792 0.690 1.3178 2.507 -0.992 -0.918 -0.761 15.690 -0.952 -1.240 Skewness 0.173 -0.183 -1.042 -0.831 0.978 0.283 0.149 0.451 -3.800 0.408 0.0302 Minimum value 0.203 73.31 31.00 65.54 0.68 0.073 0.264 11.00 0.00 Maximum value 36.93 133.44 63.00 6.069 148.47 5.667 0.628 1.173 15.00 69.28 Confidence level (95%) 4.974 8.460 3.993 0.511 8.563 0.698 0.076 0.131 2.029 0.616 11.193 BG11 6.224 111.654 63.55 4.159 93.327 5.041 0.466 1.270 28.45 13.2 22.519 2.928 114.505 66.5 4.310 91.98 4.406 0.457 1.196 7.007 19.065 15.275 1.513 10.467 1.945 0.183 0.382 6.724 27.419 49.105 363.491 233.313 2.290 109.562 3.785 0.033 0.146 45.208 1.432 751.812 -1.3502 1.111 -1.002 0.273 -0.131 -1.063 -0.787 5.783 19.624 -0.063 -1.538 0.554 -0.107 -0.605 -0.383 0.198 0.211 0.326 2.012 -4.412 0.803 0.570 0.000 66.66 34.00 0.867 73.43 1.881 0.195 0.77 12.00 18.5 154.78 80.00 7.228 116.02 8.365 0.818 2.534 16.00 68.52 3.280 8.923 7.149 0.708 4.899 0.911 0.086 0.179 3.147 0.560 12.833 FRH2 15.096 99.766 53.65 3.500 103.512 3.120 0.311 0.894 29.35 12.05 28.294 17.6 97.415 54.00 3.524 101.105 2.748 0.279 0.879 29.00 23.865 8.614 14.739 10.801 0.932 20.961 1.433 0.119 0.305 1.226 1.356 21.749 74.196 217.230 116.661 0.869 439.382 2.053 0.014 0.093 1.503 1.839 473.036 -0.248 -0.754 -0.648 -0.564 2.180 1.601 2.307 0.604 -0.326 -0.476 -0.504 -0.0325 0.048 -0.178 1.377 1.061 0.967 0.005 0.463 0.523 67.7 37.00 2.013 1.445 0.141 0.39 27.00 10.00 129.18 73.00 5.216 143.61 7.157 0.64 1.761 32.00 68.94 4.0313 6.898 5.055 0.436 9.810 0.671 0.056 0.143 0.574 0.635 10.179 Table 1: Data of phenotypic variables in groups of Arabidopsis thaliana plants inoculated with Bauveria bassiana BG11 and FRh2 strains Figure 2: Polar coordinates of total variables analysed to discriminate between control vs BG11 for phenotypical traits in groups of Arabidopsis thaliana Col-1 plants inoculated with BG11 and FRh2 Beauveria bassiana strains Table B. Dunnett’s test in groups of Arabidopsis thaliana Col-1 plants inoculated with BG11 and FRh2 Beauveria bassiana strains after β Bonferroni correction (0.0051) Conclusions Beauveria bassiana BG11 and FRh2 were recovered as endophyte from Arabidopsis thaliana Col-1 plant tissue and detected by qPCR after inoculation by dip root method. Each strain of Beauveria bassiana interacted differently with the Arabidopsis thaliana Col-1 plants. The endophytic B. bassiana did not have any antagonistic activity on the performance of P. xylostella and M. persicae. References Akello, J., & Sikora, R. (2012). Systemic acropedal influence of endophyte seed treatment on Acyrthosiphon pisum and Aphis fabae offspring development and reproductive fitness. Biological Control, 61(3), 215-221 Boyes D., Zayed A., Ascenzi R., McCaskill A., Hoffman N., Davis K., Görlach J. (2001). Growth stage-based phenotypic analysis of Arabidopsis: A model for high throughput functional genomics in plants. 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