Effects of four soil-originated Bacillus spp. on the great spruce bark beetle, Dendroctonus micans (Kugelann) (Coleoptera: Curculionidae, Scolytinae)

Microbial pest control agents (MPCAs), especially the products of different Bacillus thuringiensis (Bt) subspecies, are utilized more and more in pest management programs to combat against the larvae of certain insect vectors of agricultural crops and nuisance pests of humans (Miller 1990). An eco-friendly substitute to pesticides is provided by biological control agents to treat plant diseases. However, producers persist in utilizing chemical control rather than the alternative provided by biological control. The ignorance on this matter generally leads to the collapse of a biocontrol agent (Ardakani et al. 2010). Biocontrol can be achieved as long as the biological environment where the agent will be used is well known and the know-how of the production of a reliable and resistant formulation is available (Emmert and Handelsman 1999). Some Gram-positive bacteria are superior to their Gram-negative counterparts in terms of their natural formulation, the spore (Charles et al. 2008). Bt and its close relatives are a facultative anaerobic, Gram-positive bacterium comprising idiosyncratic protein inclusions contiguous to the endospore. The parasporal inclusions are composed of various insecticidal crystal proteins (ICP) (Van Frankenhuyzen 2009), which determine the shape of the crystals (Otieno 2010).

As its range extended from east to west progressively, the great European spruce bark beetle Dendroctonus micans (Kugelann) (Coleoptera: Curculionidae, Scolytinae) headed from Georgia to Turkey. The first report on its presence in Turkey came in 1966 (Yaman and Radek 2008). In spite of the comprehensive and costly endeavors to control the beetle, it continues to damage the economy of Turkey. In more than three decades, approximately 250.000 ha have adversely been impacted and no fewer than ten million spruce trees have been killed (Yaman and Radek 2008). D. micans is a notable defoliator of species of pine trees in Turkey and in the rest of the Mediterranean (Yaman and Radek 2008). It is also found in other countries, which have spent a great deal of money for controlling it (Grégoire 1984; Yaman et al. 2010).

It has been reported that Bt subspecies are quite unique in terms of their potential for Coleoptera, Diptera, and Lepidoptera, where they are not directly toxic to non-target arthropods (Miller 1990). Giving rise to little tree mortality, D. micans is typically observed at low levels within its natural range. Still, outbreaks which lead to extensive tree mortality are observed every now and then. The majority of the outbreaks take place along the front edge of the geographic range of D. micans and not within the inner part of the range. Trees are killed on account of the girdling action of larval feeding, which may occur during a few years. D. micans enlarged its range into Europe (France and the UK) and into southwestern Asia in the late 1900s, and therefore, the outbreaks took place on no less than 200,000 ha of spruce forests (Vakula et al. 2016).

A pilot study on pine trees was performed to analyze the body pathogens and to determine the most aggressive one on the holes in the pine tree body of young and old plants.

Four Bacillus species and two other species were isolated from the soil and identified as Bacillus mycoides, B. cereus, B. thuringiensis, Paenibacillus validus, B. atrophaeus, and Arthrobacter globiformis. Bacterial isolates were identified using fatty acid profiles, bon substrate utilization footprints, and then VITEK. The ability of Bacillus isolates to produce amylase, lipase/esterase, chitinase, xylanase, pectinase, protease, and cellulase extracellular enzyme was qualitatively investigated. The results of the enzyme activity outside the cell of the isolates of Bacillus isolates are given in Table 1. Effects of all isolated bacteria were tested on the adults and larvae of D. micans. Laboratory experiments conducted to demonstrate potential of these isolates indicated that they all had, more or less, an insecticidal impact on the larvae and adults of the bark beetle. B. mycoides had mortality rates of 60 and 50% on the larvae and adults of D. micans, respectively. A novel strain of B. mycoides strain AQ 726 that generates a metabolite pesticidal activity was observed by Heins et al. (1999) who developed a technique to restore or preserve a plant from such insect infestations as corn rootworm. The technique involved the application of a fair quantity of novel metabolite-producing bacterial strain to the plant or its environment. This bacterial strain was a supernatant comprising a metabolite acquired from a whole broth culture of the strain or the metabolite itself. B. cereus had 40 and 30% mortality on the larvae and adults, respectively. B. cereus was also accepted as an insect pathogen. It was observed that B. cereus secreted proteins that immobilize and kill cockroaches, Blattela germanica, upon injection (Man et al. 2013). The highest effect was determined with B. thuringiensis on the larvae and adults of the bark beetle. This bacterium showed 80 to 70% mortality rate on the larvae and adults of D. micans, respectively. The findings of this study showed that the larvae were more vulnerable to the isolated bacteria than the adults (Fig. 1). In the literature, few bacteria other than B. thuringiensis Berliner are lethal to the Colorado potato beetle, Leptinotarsa decemlineata (Say), a major pest of potatoes and eggplant (Martin et al. 2004). In this study, four lethal Bacillus species were found against the bark beetle, while some of which were lethal to other insects as well.

Name of bacteria	Gram stain	Spore	Shape of bacteria	Shape of colony	Colony color	Amylase/chitinase	Tween 20/Tween 80	Tributhrin Rho. B agar	Xylanase	Pectinase 1/pectinase 2	Protease/cellulase
Bacillus mycoides	+	+	Rod-shaped bacteria, non-motile (Bacillus)	Fringed, filaments, spiral pattern	White, dirty	−/−	NT	NT	NT	NT	NT
Bacillus cereus	−	−	Rod-shaped (Bacillus) motile	Convex, the edges are indented, round	White and glistening	−/−	+/+	+/+	+	*/++	+/+
Bacillus thuringiensis	−	−	Bacillus	Smooth-round	Gray	+/+	+/−	+/−	−	*/−	+/+
Bacillus atrophaeus	+	−	Bacillus non-motile	Smooth-round	Grayish white, transparent	NT	NT	NT	NT	NT	NT
Paenibacillus validus	+	−	Coccoid	Smooth or round slightly convex	Very transparent with a milky white	NT	NT	NT	NT	NT	NT
Arthrobacter globiformis	+	−	Coccus rods	Smooth or round convex	Pale cream/pale orange	NT	NT	NT	NT	NT	NT

−, zone diameter; +: < 10 mm, 10 mm ≤ ++ ≤ 20 mm, > 20 mm

NT No experiment

*Bacteria have not grown

No studies addressing entomopathogenic bacteria in bark beetles have been found in the literature (Yaman et al. 2010). Wegensteiner (2004) reviewed the bacteria of bark beetles and provided several species recorded by different scientists. A few of these studies are on D. micans. Imnadze (1978) pointed out the presence of B. thuringiensis in D. micans. Yılmaz et al. (2007) isolated seven bacteria from this pest and compared the isolates with the bacteria recorded from bark beetles, where it was found that only two of them had been isolated from those insects before. P. validus, B. atrophaeus, and A. globiformis exhibited the same effect with the mortality rates of 30 and 20% on the larvae and adults of D. micans, respectively. Being a genus of facultative anaerobic, endospore-forming bacteria, Paenibacillus was initially categorized as belonging to the genus Bacillus, but recategorized as a different genus in 1993 (Zhao et al. 2012). The bacteria of this genus are commonly observed in various media (Simonová et al. 2015). Paenibacillus polymyxa (Bacillus polymyxa) is a Gram-positive bacterium, which is also able to fix nitrogen (Vilinska et al. 2008). Being Gram-positive, B. atrophaeus is a biological indicator for sterilization (Christensen and Kristensen 1979). Luo et al. (2012) reported that Bacillus atrophaeus CPB072 had poisoning and controlling effects on potato beetles. It can grow and massively produce spores in a Luria-Bertani culturing medium, at 30 °C and pH 7.0. Inoculation concentration > 10⁸/ml was the optimum condition for fungal infection pathogenicity.

Pesson et al. (1955) reported that Aerobacter scolyti and Escherichia klebsiellaeformis led to adequate mortality rates in Scolytus multistriatus within 72 h. The number of studies on the bacterial pathogens of D. micans is scant. Yılmaz et al. (2007) isolated seven bacteria on D. micans; however, they failed to test their effects on the pest. Conducting experimental infections of isolated organisms from insects is crucial to substantiate the potential of their use as biological control agents.

The findings of the experimental infections are encouraging. However, they are limited to laboratory scale. It is also necessary to conduct field treatments on these isolates. We had some difficulties for keeping the larvae and adults of D. micans in test boxes for a long-term trials. For this reason, probably, Yılmaz et al. (2007) failed to test the effects of these isolates on D. micans. Tonka et al. (2008) accomplished to analyze the laboratory management of entomopoxvirus in Ips typograhus and found that 595 out of 1142 offered beetles were infected by the entomopoxvirus. They tested this virus in a laboratory for several weeks.

Approximately 50 isolates of bacteria were isolated from 156 soil samples. Identification was carried out, and six bacterial species were tested in the present study. FAME analysis and VITEK results are too long to include them in this study. It was demonstrated a number of bacterial species that are lethal to D. micans. Most of the biological control studies on this pest have concentrated on the predatory beetles but also reported a significance potential effect of these bacterial species on the larvae and adults of D. micans. In conclusion, these bacteria are efficient biological control agents to combat D. micans.