Date Published: February 1, 2019
Publisher: Public Library of Science
Author(s): Sumer Alali, Valeria Mereghetti, Franco Faoro, Stefano Bocchi, Fawaz Al Azmeh, Matteo Montagna, Yulin Gao.
The use of Beauveria bassiana in biological control of agricultural pests is mainly hampered by environmental factors, such as elevated temperatures and low humidity. These limitations, further amplified in a global warming scenario, could nullify biological control strategies based on this fungus. The identification of thermotolerant B. bassiana isolates represents a possible strategy to overcome this problem. In this study, in order to maximize the probability in the isolation of thermotolerant B. bassiana, soil samples and infected insects were collected in warm areas of Syria. The obtained fungal isolates were tested for different biological parameters (i.e., growth rate, sporulation and spore germination) at growing temperatures ranging from 20°C to 35°C. Among these isolates (eight from insects and 11 from soil samples), the five with the highest growth rate, spore production and germination at 30°C were tested for their entomopathogenicity through in vivo assays on Ephestia kuehniella larvae. Insect mortality induced by the five isolates ranged from 31% to 100%. Two isolates, one from Phyllognathus excavatus and one from soil, caused 50% of the larval mortality in less than four days, reaching values exceeding 92% in ten days. These two isolates were molecularly identified as B. bassiana sensu stricto by using three markers (i.e., ITS, Bloc and EF1-α). Considering these promising results, further studies are ongoing, testing their efficiency in field conditions as control agents for agricultural insect pests in Mediterranean and Subtropical regions.
Global climate change poses new challenges to agricultural production , increasing crops’ vulnerability to plant diseases and pests mainly as a consequence of physiological stresses , and the expansion of insects and diseases into higher latitudes . The introduction of new pests, aside from endangering food security, requires the adoption of new strategies for their control. This phenomenon particularly affects temperate countries, where the expansion of species range due to climate change has been well documented . In particular, it has been estimated that insects and diseases are expanding towards the poles at 7 km per year . In agriculture, adaptive strategies have already been used to improve the capability of plants to face increasing abiotic stresses and to optimize growth and phenology in relation to new seasonal variations [6–7]. In pest control, the keystone adaptive strategy relies on integrated pest management (IPM), which depends upon different integrated practices starting from an accurate estimation of the pest abundance based on monitoring and working towards the adoption of biological control strategies to reduce of conventional pesticide application . Mycoinsecticides, mainly consisting of the fungal pathogen Metarhizium anisopliae (Metschn.) Sorokın and Beauveria bassiana Balsam are a fundamental component of these biological control strategies [9,10]. Even though these fungi have already been successfully adopted in large-scale applications (e.g., for the control of the Sahelian grasshopper Oedaleus senegalensis Krauss, 1877  and the Masson’s pine caterpillar Dendrolimus punctatus Walker, 1855 ) and their use is increasing yearly (20–44%) in Europe and North America , they suffer major limitations. In fact, different abiotic factors, such as UV radiation, low humidity and high temperature, hamper fungal biological activity and, in turn, their effectiveness as biocontrol agents [14–15]. For example, the optimal growth temperature of B. bassiana is between 25°C and 28°C , though it has been demonstrated that some isolates are able to grow at a higher temperature (~30°C) with highly reduced activity and may not survive at 34°C [17,18]. To overcome this limitation, a possible strategy has been developed, which relies on genetic improvement of the fungus in terms of its tolerance to oxidative stress was developed [19,20]; however, this approach suffers regulatory and societal limitations related to the use of genetically modified organisms . A possible alternative strategy consists of mass screening of fungal isolates for the target phenotype, a successful strategy in the case of UV tolerance .
In this study, all B. bassiana isolates germinated and grew at 20°C, 25°C and 30°C, but not at 35°C, as expected. Five B. bassiana isolates, namely bbpp1, bbph2, bbL4, bbph13 and bbbm14, performed well in the tested biological traits, also growing at 30°C (S5 Table). It is worth noting that four out of the five isolates were collected in the hottest areas. The only exception is represented by isolate bbL4, obtained from a site with a lower temperature (27°C). The results of this study support the existence of a relationship between the termotholerance ability of the isolates and their geographical origin, even if this is still under debate [32,51]. In terms of pathogenicity, the five selected isolates induced a differential mortality on the model insect (i.e., E. kuehniella), with values ranging from 31% to 100% (Table 1). Isolates bbph2, bbL4 and bbph13 achieved more than 75% mortality, whereas isolates bbph2 and bbL4 showed the highest values of FDI within the same time period (2.73±0.27, 2.87±0.03, respectively), which highlights the difference in pathogenicity levels between the isolates . The achieved values of pathogenicity further support adaptation of the isolates to growth at a relatively high temperature. In addition, the isolates bbph2 and bbL4 possessed a low value of LC50 (1.65 × 104 and 2.01 × 104, respectively), which are in a range of values considered effective on lepidopteran larvae as E. kuehniella and Plodia interpunctella [53,54]. The values of LT50 obtained for these two isolates (less than four days) confirm their aggressivity, according with the findings of Sosa-Gomez . In addition, the two isolates possessed significantly higher values of chitinase activity among those tested (Table 2), a feature correlated with their entomopathogenicity . Multilocus-based molecular identification of isolates bbph2 and bbL4 confirmed the morphology-based identification as B. bassiana s.s. Phylogenetic analyses, performed on each single marker and on the concatenated dataset, attributed the bbph2 (isolated from the beetle P. excavatus) and bbL4 (isolated from a soil sample) isolates to two separate B. bassiana clades: the first, with B. bassiana isolated from beetles collected in the USA (L. decemlineata and A. grandis); the second, sister of B. bassiana isolated from a weevil collected in Morocco. Even considering the limited sample size, the achieved results apparently indicate the absence of a correlation in terms of geographical origin of the isolates and host taxonomy. For example, isolates obtained from the same geographical area (Morocco) and from the same host (Sitona discoideus) were grouped into two separate and well-supported clades (Fig 4, S4 Table).