Beauveria Bassiana as Fungal Endophyte for the Potential Control of the Potato Tuber Moth Phthorimaea Operculella on Potatoes

22 The potato tuber moth, Phthorimaea operculella , is the most damaging potato pest in the world and is 23 difficult to control as the larvae are internal feeders in the foliage or tubers. Entomopathogenic fungi 24 (EPF) which colonize plants as endophytes, have lethal and sublethal pathological effects on insect 25 pests. Experiments showed that Beauveria bassiana colonized the aerial parts of potato plants 26 endophytically after inoculation through soil drenching. The colonization rate reached 100% for both 27 upper and lower foliage parts one day after inoculation, and endophytic B. bassiana remained present 28 for more than 21-day post inoculation. Mortality experiments indicated that B. bassiana and B. 29 bassiana -inoculated potato plants were pathogenic against 2 nd instar larvae of P. operculell a. 30 Development experiments showed that the weight of P. operculell a pupae reared on B. 31 bassiana -colonized potato leaves (4.25 mg) was significantly lighter than of those reared on 32 uninoculated control plants (8.89 mg). Sublethal experiments indicated that B. bassiana negatively 33 affected the growth, development and reproduction of P. operculella . Compared to newly eclosed 34 larvae fed on control plants, those fed on B. bassiana -inoculated plants had significantly lower survival, 35 with only 17.8% developing to the adult stage. Oviposition of P. operculell a females reared on B. 36 bassiana endophytically-colonized plants was significantly lower (35 eggs/per female) than of those 37 reared on uninoculated plants (115 eggs/per female). This study demonstrates that endophytic B. 38 bassiana can be a potential biological agent for the control and management of P. operculell a.


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Larvae of this species mine leaves, stems, petioles, and excavate tunnels through potato tubers (Gao

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The objectives of this study were therefore to determine whether the B. bassiana isolate, 110 GZGY-1-3, could colonize potato plants endophytically using the soil drench method and to assess its 111 pathogenicity as an endophyte, as well as to determine its sublethal effects on P. operculella.    This was followed by immersion in 1.5% sodium hypochlorite for 3 min, rinsing with sterile distilled 150 water three times, and then drying on sterile paper towel for 30 s. They were finally cut into 1 × 1 cm 151 pieces under a laminar flow hood before placing them on PDA plate. In addition, water from the last 152 rinse was plated out to assess the reliability of the surface sterilization procedure (Bamisile et al.

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Only the presence of B. bassiana endophyte that was inoculated was scored. Fungal colonies from 158 surface-sterilized parts were characterized only when mycelia grew at the edge of leaf segments from 159 internal tissues. To confirm whether the growing endophytes were the ones initially inoculated, slides 160 prepared from the mother plates were used for comparison and morphological identification. The data 161 was expressed as percent colonization. We separately observed colonization at 1, 4, 7, 14 and 21d 162 after the soil drenching treatment. The experiments were repeated five times using eighteen plants for 163 each treatment (Table 1). Independent batches of plants and B. bassiana were used in each treatment.

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We adopted the leaf-disc assay method to determine whether B. bassiana and B. bassiana-inoculated 166 potato plants was pathogenic against P. operculella larvae. In these bioassays, four treatments were

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The bioassays were carried out in an artificial environmental chamber (MLR-351H, SANYO 182 Electric Co., Ltd., Moriguchi City, Osaka, Japan) under controlled conditions of 26 ± 1 ℃, 12:12 h 183 L:D, and 70% ± 10% RH. Mortality of P. operculella was recorded daily up to 13 days after infestation. Each treatment was replicated twice, with twenty-four 2 nd instars per replicate. The 185 bioassays were repeated five times using independent batches of plants and larvae (Table 1)

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One-way analysis of variance (ANOVA) and Tukey's HSD test were used to assess virulence 216 bioassays (mortality and LT50). Mortality data were corrected using Abbott's correction (Abbott 1925).

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The median lethal times (LT50) were determined by probit regression. Pupal weight was compared by  from the entire cohort. The age-specific survival rate (lx) was then calculated as follows: The intrinsic rate of increase (r) was estimated using the Euler-Lotka formula, with the age indexed

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However, mortality was significantly greater for larvae exposed to conidia than for larvae exposed to 253 plant tissue inoculated by B. bassiana. At 3dpi, the corrected mortality was 51.22% for larvae directly 254 exposed to conidia and 35.28% for larvae exposed to plant tissue endophytically inoculated by B.
255 bassiana ( Fig. 2A). At 7dpi, the corrected mortality for larvae directly exposed to conidia was 88.35% 256 and 63.59% for larvae exposed to plant tissue inoculated by B. bassiana. At 8dpi, the corrected 257 mortality of P. operculella exposed directly to conidial suspension reached 100%, which was 258 significantly higher than that of larvae that fed on B. bassiana-inoculated potato plants (68.16%; t =

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-30.425, df = 9, P < 0.001). There was a synergistic effect from the combination of the two B.

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The results also indicated that endophytic B. bassiana were virulent to P. operculella larvae but 263 acted at a slower rate. The LT50 for P. operculella exposed to endophytic B. bassiana was 5.57 d,

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The curves of the age-stage survival rate (sxj) show the probability of an individual surviving to age 285 x and developing to stage j (Fig. 5A, 5B) (Table 3). Statistical analysis showed that the r for P.   there was also direct pathogenicity to P. operculella. The mechanism by which endophytic fungi 342 interact with insects is still unclear but the involvement of metabolites/antibiosis produced by the 343 fungi can be speculated (Vega 2018