Can We Use Entomopathogenic Fungi As Endophytes For Biological Control Of The Horse-Chestnut Leaf Miner?

The horse-chestnut leaf miner, Cameraria ohridella (Lepidoptera, Gracillariidae), is a major pest of horse-chestnut trees (Aesculus hippocastanum) in Europe. This little moth (only 5mm long) was first observed in Macedonia in 1984, however, its Balkan origin has been yet under scientific discussion. Since the first observation, it has colonized large areas of Europe and is still spreading.

It was listed among 100 of the worst invasive species in Europe (Augustin, 2009) and therefore its biology and ways for effective management have been thoroughly studied (Gilbert et al., 2003; Baraniak et al., 2005; Kenis et al., 2005). At high densities, the moth completely defoliates trees already in summer. The highly-visible leaf damage is caused by feeding of larvae between the upper and lower leaf epidermis. The damage is particularly spectacular in cities where horse-chestnut trees are abundant and the defoliated trees in parks and gardens in early summer raises public concern.

The leaf miner overwinters in leaf litters as pupae, and adults appear during May. Females lay eggs on the upper leaf side and larvae of new generation hatch within two weeks. They bore through the leaf epidermis into assimilation parenchyma where they mine internal tissue resulting in typical leaf damage. The larvae reach a pupal stage in the mines and emerging adults of the second generation leave the mines and reproduce. In a season, up to 2‒4 generations can develop (Girardoz et al., 2007).

Up until now, attempts to effectively control the leaf miner have failed. Control strategies have been mainly focused on mechanical measures (e.g. removal of leaf litter) (Kehrli and Bacher, 2003), pesticide application (Kuldová et al., 2007), or sex pheromone techniques (Sukatova et al., 2011). Unfortunately, these methods are either expensive, short-term, or ecologically questionable, therefore an alternative control approach with a use of fungal pathogens (entomopathogenic fungi) is also considered (Schemmer et al., 2016).

Entomopathogenic fungi are natural parasites of insects that help in the prevention of formation of insect population outbreaks. These fungi can spread fast among insect populations via spores and infect hosts by penetrating the skin with germinating hyphae. Different entomopathogenic fungi have been tested against various insect pests and several have been successfully licensed as mycopesticides (Faria and Wraight, 2007).

Activity of entomopathogenic fungi in the leaf miner populations has already been studied in fields and tested in the laboratory. Although virulent strains of fungi Beauveria bassiana and B. pseudobassiana were identified against the leaf miner pupae (Schemmer et al., 2016), in field environments, where the fungi were applied as spore sprays to leaf litters against hibernating pupae, only suboptimal results were obtained.

A number of studies demonstrate that efficacy of these fungi as microbial insecticides are affected by adverse abiotic factors including temperature, humidity, solar radiation, etc. With the aim to eliminate these ambient factors, attempts to use the fungi as endophytes in plants have been tested as a novel control strategy (Jaber and Ownley, 2017). Generally, endophytes are defined as microorganisms existing inside healthy plant tissues without causing any apparent symptoms or harm to their hosts (Wilson, 1995). They are known to fulfill a variety of functions in plants that may benefit their hosts including a protection against insect pests.

It is known that certain entomopathogenic fungi can form an endophytic association with plants. B. bassiana, the most common insect pathogen, has been found naturally as an endophyte in several plant species and has been artificially introduced into many others. There is accumulating evidence that endophytic B. bassiana can reduce a pest damage to plants indirectly by inhibiting insect development and reproduction or directly by causing insect mycosis resulting in mortality. Entomopathogenic fungi as endophytes can thus be a prospective approach to plant protection systems, reducing the risk of pesticide use and inducing resistance against insect pests (Vidal and Jaber, 2015).

Endophytic association of entomopathogenic fungi with horse-chestnut trees and their role as active protective agents in leaf tissue has been recently studied. Laboratory bioassays were conducted to determine if Beauveria bassiana and B. pseudobassiana can be artificially established as endophytes in horse-chestnut leaves and investigate the effects of endophytic Beauveria on the horse-chestnut leaf miner. Two strains of B. bassiana and one strain of B. pseudobassiana, which had demonstrated high virulence to leaf miner pupae, successfully colonized horse-chestnut leaves after a topical treatment with spore suspensions. The colonization rate of leaves reached high values and although the level of colonization decreased over time the fungi were still present in leaf tissue two months post-inoculation.

Colonized leaves were artificially infested by the horse-chestnut leaf miner and it was demonstrated that damage by larvae was significantly lower in Beauveria-colonized leaves compared to non-colonized plants. The damaged leaf area of colonized leaves was even 5-times smaller compared to non-colonized leaves. Although the leaf miner larvae could develop in Beauveria-colonized leaves, their survivorship was reduced by up to 57%. Besides the effect on the leaf miner survivorship, a size of pupae from pest population developed in colonized leaves was also affected. Pupae that completed their development in colonized leaves had significantly reduced size and weight. The dramatically lower damage on colonized leaves, the reduction in leaf miner survival and the size of pupae indicate that Beauveria strains colonizing leaves effectively exhibited insecticidal effect when growing inside the horse-chestnut tissue.

An incorporation of entomopathogenic endophytes into trophic interactions between the leaf miner and horse-chestnut trees demonstrates their potential for use in insect pest management; however, a further investigation, which should focus on an application technology in the field, long-term establishment of endophytes in trees, and side effects on non-target organisms, is necessary.

These findings are described in the article entitled In planta bioassay on the effects of endophytic Beauveria strains against larvae of horse‐chestnut leaf miner (Cameraria ohridella), recently published in the journal Biological Control. This work was supported by the Slovak Grant Agency VEGA (grant no. 2/0025/15) and conducted by Marek Barta from the Institute of Forest Ecology of the Slovak Academy of Sciences.

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