Efficacy of Beauveria bassiana and Mechanical Traps for the Control of Aclees taiwanensis (Coleoptera: Curculionidae) in Fig Plants

Abstract

The black weevil Aclees taiwanensis Kôno, 1933 (Coleoptera: Curculionidae) is a xylophagous insect native to Southeast Asia and introduced to Italy in 2005. Here, the species completes its entire life cycle on Ficus carica L., causing economic damage and leading to the plants’ death. Nowadays, there are no insecticides registered for its control. In this study, a commercial product based on Beauveria bassiana, Naturalis^®, was associated with Rincotrap^® tissue. Semi-field trials were carried out on fig seedlings by comparing two different concentrations (3% and 10%) of this entomopathogenic fungus distributed in suspension on Rincotrap^® bands. The results showed that the fungal product had a biocidal effect in both treatments. Afterward, trials were conducted in a fig orchard in order to verify the effectiveness of this association in the field. The addition of Naturalis^® at a 3% concentration to Rincotrap^® bands did not alter the number of adults captured. The total mortality was 43.7% and 23.8%, respectively, in the treated and control plots. Among the total number of dead adults, only the mortality rates of B. bassiana exceeded 70% in both plots. Further long-term studies in several environments are necessary to improve this technique and create an integrated control system for the black fig weevil.

1. Introduction

The fig tree (Ficus carica L.) represents one of the oldest crops in the Mediterranean area. In Italy, the species has been cultivated since at least the 8th century BC. The Italian regions with the greatest production vocation are Apulia, Campania, and Calabria; a significant production also comes from Abruzzo, Sicily, and Lazio. Apulia also provides the greatest production of dried figs [1]. The fig has been and remains widespread in mixed cultivation with other fruit or herbaceous crops; however, recently, despite the general decline in cultivated areas, the number of intensive systems for the production of figs is increasing. There are over 600 varieties/genotypes of F. carica reported and cultivated, many with local diffusion, which is evidence of a rich germplasm [2]. Therefore, in light of the climate change which impacts fruit cultivation in the Mediterranean region, the fig can play a valuable role. The fig has always been considered a very rustic species; it is adaptable to many marginal environments and requires little attention, even from a phytosanitary point of view. Unfortunately, in recent years, due to the introduction of exotic species that increasingly create phytosanitary problems [3], the fig has also started to have enemies that can potentially damage its production and survival. Aclees taiwanensis Kôno, 1933 (Coleoptera: Curculionidae), the black fig weevil, native to Southeast Asia, was first reported in Italy in 2005, with an incorrect taxonomic identification as A. cribratus and subsequently as A. cf. sp. foveatus [4,5]. The genus Aclees Schoenherr 1835 is composed of over 30 species distributed in tropical Africa and Southeast Asia, but the taxonomy of the genus was still extremely confused. Furthermore, most of the species are morphologically very similar and this has generated confusion in the identification of samples preserved in museums and collections. Only in 2020, Meregalli and coauthors [6] correctly revised the taxonomic identification, referring to the species introduced in Italy and France as A. taiwanensis. Currently, the weevil is widespread in several regions of Italy (Abruzzo, Apulia, Emilia Romagna, Friuli-Venezia Giulia, Lazio, Liguria, Lombardy, Marche, Tuscany, Umbria, Veneto, and Sicily) [7,8,9,10]; since 2020, it has also been detected in France, in the area around the Sauvebonne Valley [11], and in South Korea [12]. The host plants of this species belong to the Ficus genus, and the majority of the economic damage reported in Italy is specifically regarding the common fig F. carica [7]. The damage to the plants is caused by the xylophagous larvae that, during their development, dig tunnels inside the trunk and, to a lesser extent, by the adults that feed on leaves and ripening fruits. Due to the ethology of the species, which spends a large part of its life cycle within the tissues of the plant, the infested plants show symptoms only at an advanced stage of infestation, thus making the care and recovery of the plant very difficult. Currently, no fig cultivars resistant to the phytophagous attacks have been found [4]. The weevil can also attack other ornamental plants of the Ficus genus, such as F. pandurata, F. benjamina, and F. macrocarpa [7], thus increasing its chances of spreading; however, further research is needed. The lack of national and international regulation and the rapid spread of the species in several production areas has led us to assume that this insect could soon become an important pest for fig trees in the Mediterranean area.

The biology and ethology of A. taiwanensis have only recently been studied [5,7,13]. No registered products are available for its control. The control strategies tested in the past have not proven to be effective. The only stage of the insect that can be affected by insecticidal products is the adult stage since the larvae grow in the trunk. Tests conducted so far using various synthetic active ingredients with a contact action (e.g., pyrethroids) have not given the desired results [14]. Other studies regarding substances with masking effects on the plants, such as aluminosilicate minerals (e.g., bentonite, zeolite), displayed quite promising results, but they should be used in an integrated pest management (IPM) strategy [15]. Preliminary laboratory tests with Spinosad (produced by the bacterium Saccharopolispora spinosa) at two different concentrations have given results of some interest [16], reaching an adult mortality rate of 100% after 72 h, but at the moment this product is not registered for fig crops. Very promising results were obtained in a preliminary laboratory study with plant extracts, such as olive oil and onion treatments: a mortality rate of 100% was reached after 24 h of olive oil treatment, while after only 1 h, the onion oil extract led to an 80% mortality rate [17]. Concerning biological control agents, the only natural enemy found in Italy is a strain of the entomopathogenic fungus Beauveria bassiana [18]. Laboratory and semi-field tests with Naturalis^®, a commercial product based on B. bassiana, have shown noteworthy results. In the laboratory trials, adult mortality reached 100% with Median Lethal Time (LT50) in 8.5 days. In the semi-field test, two different doses of commercial product were sprayed on the whole plant. The highest mortality rate for adults was registered after 35 days from the treatment [18,19]. Concerning mechanical methods, bands of synthetic material of inert fibre (Rincotrap^®) showed good results for monitoring/capture of adults in the field [7,20].

For these reasons, the main aim of this work is to verify the effectiveness of the association of the commercial product based on B. bassiana (Naturalis^®) with the Rincotrap^® bands to contain A. taiwanensis adult populations in infested fig crops.

2. Materials and Methods

2.1. Semi-Field Trials in Entomological Cages

Two-year-old fig seedlings of the cv Dottato, grown in 30 cm pots, were placed in BugDorm-2 Insect Rearing Tent (75 × 75 × 115 cm) (BugDorm, NHBS Ltd., Totnes, UK) and kept in a greenhouse (25 ± 5 °C; UR 60%). Rincotrap^® bands (Greenagri S.r.l., Bari, Italy) (16 × 5 cm) soaked with 10 mL suspensions of Naturalis^® (CBC(Europe) S.r.l., Biogard division, Grassobbio (BG), Italy) at 3% and 10%, were positioned at the base of the trunks of the plants. To improve the physical characteristics of the suspension, 0.02% Tween 80^® was added; in fact, the surfactant used is recognized as a critical component in assisting the conidia of a fungus to germinate and infect the target organism [21]. In the control plants, the bands were soaked only with tap water and 0.02% Tween 80^®. Three plants (one per cage) were considered for each treatment, 3%, 10%, and control, for a total of nine plants. The plants were watered once a week to soil capacity. Four males and four females of A. taiwanensis were placed in each cage for a total of 24 adults per treatment (n = 72 total adults). A second treatment with Naturalis^® at the same concentration was repeated, according to laboratory trials performed by Benvenuti et al. [22], after two weeks, since the fungus germination reduced over time when distributed on the Rincotrap^® bands. Checks were performed once a week, up to 28 days after the second treatment. Dead individuals were collected and sterilized with 1% sodium-hypochlorite for 3 min and rinsed with sterile water [23]. Finally, all specimens were placed on moistened filter paper in Petri dishes and kept at 25 ± 1 °C to verify the presence of B. bassiana according to Llacer et al. [24]. The total mortality (number of dead adults due to mycosis and other causes) and the mortality due to Naturalis^® (number of adults killed by B. bassiana) were calculated.

2.2. Field Trials

The trials were conducted in a commercial fig orchard in the Fosdinovo area, located at 120 m a.s.l. (N 44.126661; E 9.992756; Massa-Carrara province), in Northern Tuscany (Italy) from April to June 2021. In the cultivation area, which included about 50 plants (4 × 4 m planting layout), two similar plots were selected, each consisting of ten plants of the cv Fioroni, of homogeneous age and size. Two rows of ten fig plants were used as a buffer zone between the two plots. All plants showed evident signs of attack by the black fig weevil (i.e., at the collar and at greater heights along the trunk, with the presence of fresh sawdust and early phylloptosis). In the control plot, a band made of Rincotrap^® was positioned on each of the ten plants at the collar; firmly fixed around the base of the trunk by tying, according to the protocol proposed by Farina and co-authors [7]; and wet with tap water and 0.02% Tween 80^®, according to Benvenuti et al. [22]. Since no differences were found between the 3% and 10% concentrations of Naturalis^® in the semi-field trials (see Section 3), the first one was used in the field. In the treated plot, a band made with Rincotrap^® was positioned at the collar of the ten plants as in the control plot and moistened with a suspension of B. bassiana 3%, 0.02% Tween 80^® and tap water.

Since previous studies had shown a reduction over time in the germination of the entomopathogenic fungus [22], a new 3% suspension of B. bassiana was applied after 30 days. Indeed, as reported also in Section 3 (see the second table in Section 3.2), no significant differences were found concerning spore concentration and germination between 15 and 30 days. The checks in both plots were carried out weekly during the entire period. During the surveys, the adults of A. taiwanensis captured in the bands were collected and transferred to the laboratory of CREA-DC in Florence, for the evaluation of the B. bassiana infection. Moreover, collected adults were identified and sexed: the shape and position of the last tergite of the abdomen curves downward in males but is horizontally placed in females, according to Farina et al. [7]. To verify the presence of B. bassiana, the insects of both groups were kept under observation until the end of August 2021, keeping them isolated in individual transparent PP boxes (14 × 14 × 5 cm) with perforated lids. The adults were fed regularly with fresh fig leaves and twigs, and a water supply was placed inside each box. Dead individuals were analysed as in the semi-field trials to verify the presence of B. bassiana.

Furthermore, to confirm the results obtained by Benvenuti et al. [22] and to verify the spore concentration and germination of the fungus, three portions (1 cm²) of treated Rincotrap^® tissue from three plants were picked up and washed in 1 mL of distilled water at the beginning (T0), after two weeks (T1), and one month (T2) before the second treatment.

The spore concentration in the resulting suspension was determined using haemocytometer [25]. The quantification of the germination of the fungus in the Rincotrap^® was evaluated according to the protocol described by Liu et al. [25]. The spores were observed under an optical microscope to evaluate the morphological characteristics attributable to the genus Beauveria. Samplings over time were necessary to verify the presence of the fungal inoculum in the field, because a lot of environmental factors can influence the stability of a fungal strain, which is sensitive to variations in temperature, humidity, and UV [26].

The total mortality and mortality due to Naturalis^® were evaluated as described for the semi-field trials.

2.3. Data Analysis

For the semi-field trials, total mortality, calculated with the Abbott formula, was compared by means of a contingency table and the χ² test. For the field trials, the number of catches in the two plots was evaluated, along with the mortality of the adults, and statistically compared by calculating the χ² test. Data regarding spore concentration and germination were analysed by an ANOVA test, followed by Tukey’s test, since the data were normally distributed, and statistical significance was defined as p < 0.05. All the statistical analyses were performed using Past 4.0 [27].

3. Results

3.1. Semi-Field Trials in Entomological Cages

The total mortality of adults, due to mycosis and other causes, was 71.42% in the entomological cages treated with 3% Naturalis^® and 57.14% in cages treated with 10% of the entomopathogenic fungus (Figure 1). The mortality due to B. bassiana was 72.2% at 3% and 100% at 10%.

Concerning the total mortality, both treatments were significantly different from the control (χ² = 11.894; df = 2; p = 0.002614), in particular, 3% vs. Control (χ² = 11.021; df = 1; p = 0.000901) and 10% vs. Control (χ² = 11.894; df = 1; p = 0.002614); however, the two treatments were not different from each other (χ² = 0.33566; df = 1; p = 0.56234). Concerning the mortality due to B. bassiana, no differences were found (χ² = 1.3402; df = 2; p = 0.51166).

3.2. Field Trials

A total of 42 adults (28 males and 14 females) were captured from the control plot with Rincotrap^® bands only and 32 (17 males and 15 females) in the treated plot with Rincotrap^® bands treated with Naturalis^® without significant differences (χ² = 0.29; df = 1; p = 0.5897; Figure 2).

All captured specimens (n = 74, 50 alive and 24 dead) were then kept under observation for the evaluation of B. bassiana infectivity. Of the 24 dead individuals, 17 were infected by the entomopathogenic fungus B. bassiana and, of these, only ten were collected from the treated plot and seven from the control one (

Table 1. Total number and dead of A. taiwanensis adults found in the Rincotrap^® bands and number of individuals that died due to B. bassiana infection.

Plot	Adults Captured	Total Dead Adults	Adults Killed by B. bassiana
Control	42	10	7
Treated	32	14	10
Total	74	24	17

).

The percentage of total mortality found in the treated plot was 43.74%, while that in the control plot was 23.80%, without statistical differences (χ² = 3.29; df = 1; p = 0.06). No significant difference was found regarding the percentage of mortality due to B. bassiana in the two plots (χ² = 1.350; df = 1; p = 0.2452; 70% in control plot and 71.4% in the treated one). The period between the capture and the death of the adults of A. taiwanensis due to the fungus ranges from 9 to 53 days.

Table 2. Mean and standard error of spore concentration and germination of B. bassiana in the field at the beginning (T0), after two weeks (T1), and after one month (T2). Different letters in columns indicate statistically significant differences.

	Spore Concentration	Germination
T0	1.8 × 107 ± 4.6 × 106 a	40.33 ± 2.4 a
T1	8.3 × 106 ± 1.9 × 106 a	23.33 ± 0.9 b
T2	7.4 × 106 ± 1.6 × 106 a	23.33 ± 1.20 b

shows the spore concentration and germination of B. bassiana in 24 h.

4. Discussion

The adults of A. taiwanensis are the target of the current research. Even though they have wings and can fly for a long distance, they usually move on the fig tree by walking [7]. In fact, the use of bands of inert material as mechanical traps (Rincotrap^®) (already commonly used for monitoring other species, such as Otiorhynchus spp., Curculio, and Buprestidae [28]) showed good results [20]. The insects, walking on the surface of the trunk, are captured in the fibres of the bands where they can die; however, the insects can also use them as shelter to stop or hide in without getting caught in the bands [20]. Therefore, a combination of mechanical means and biocide (Naturalis^®) could increase the effectiveness of the latter, since the infectivity of the entomopathogenic fungus could be enhanced by the insect remaining in the treated trap. Moreover, the bands can create a favourable environment for the survival of the fungus. If the insect meets the strip of Rincotrap^® treated with B. bassiana, but is not retained by the trap, it could become infected and die elsewhere, spreading the fungus into the environment, as already reported for other pests such as Rhynchophorus ferrugineus [24]. Although the distance between experimental plots was substantial (20 m) and most weevils captured were likely to have originated in the plot they were captured in, we can assume that some weevils could have entered a plot from neighbouring plots, e.g., because of inter-tree dispersal, as is also reported by Shapiro Ilan et al. (2008) [29].

The two concentrations of Naturalis^® (3% and 10%) tested in the semi-field trials determined a mortality of Aclees adults significantly different from the control, but without significant differences between the two treatments. It should be noted that in both treatments the total mortality was high, reaching 71.42% and 57.14%, respectively. Of the total dead adults, 72% (3%) and 100% (10%) developed a B. bassiana infection. Despite the effectiveness of the fungus, it is necessary to emphasise that the time that elapsed between the treatments and the death of the individuals was long compared to the reproductive biology of the weevil [7]. This was also highlighted by El Sufty et al. (2009) [30] in trials carried out against Rhynchophorus ferrugineus adults in a palm grove.

In the field, Rincotrap^® bands captured 74 adults in total. The bands designed and marketed for other species of Curculionidae (Otiorhynchus spp.) have already been used to evaluate the presence of A. taiwanensis in an open field [7,20]. Since the beginning of the trial conducted in Fosdinovo, there has been an increasing trend in total captures, with a peak at the beginning of May 2021. Then, the number of individuals caught gradually decreased. This trend agrees with previous studies on the bio-ethology of A. taiwanensis that highlighted two peaks of presences during the year: the first between the end of April and June and the second between September and October [4,7].

The total number of males collected was greater than that of females. Given that, in A. taiwanensis populations reared on an artificial diet, females represent 61% with a sex ratio equal to 1.5:1 [13], it can be hypothesized that the greater presence of males in the field could be related to a pre-copulative behaviour and not to the natural sex ratio. In other studies, during the same period, an increase in the volume of the gonads in adults of both sexes of A. taiwanensis [31] was indeed observed, which is an indication of the sexual maturity of adults and of the greater mobility of males in search of females [4].

Rincotrap^® band captures in the field were not significantly different between the two plots, treated or not with the entomopathogenic fungus (32 versus 42 adult A. taiwanensis, respectively). On the other hand, the percentage of mortality due to B. bassiana is interesting and was equal in both the control and treated plots (about 70%, a value comparable with what was observed in the semi-field test). Other studies conducted on Curculionidae showed quite similar results. In fact, Shapiro Ilan et al. (2008) [29], in field trials against the pecan weevil adults, registered a mortality rate of 78.3% after B. bassiana treatment at the trunk of the plants, while Perault et al. (2009) [32] obtained a mortality rate of 77% of adults, using a commercial product based on B. bassiana against the plum curculio. The similar percentage of infected adults captured in the two plots allows us to hypothesize that the percolation and runoff of the abundant rains, registered during the experimentation period, led to the concentration of the spore-based suspension of B. bassiana at the base of trees under the bands. Here, the insects were able to contaminate each other without being captured and then move independently into the plot not treated with the fungus. The dissemination of spores has been verified by other authors who have even exploited this technique by using attractive traps containing the spores of B. bassiana to spread them in the environment [33,34]. However, we cannot exclude the idea that B. bassiana could also be present in the environment (see Gargani et al., 2016) [18], but the high percentage of infected adults in the control plot makes the first hypothesis more plausible. Regarding the germination of the fungus over time, as already shown by Benvenuti et al. [22], there is a reduction after one month; therefore, it needs a second treatment to maintain its efficiency. The Rincotrap^® bands can protect the fungus from environmental factors such as UV and drought that can hamper the conidia germination [35]. On the contrary, as reported by Sabbahi et al. (2009) [36], the presence of viable B. bassiana conidia declined after six days from a treatment on strawberry leaves, since the fungus was directly exposed to environmental stressors. However, the optimal number and frequency of application may depend on several factors, such as the above-described environmental conditions, but also the pest density and the formulation of the product.

The observations made did not allow an estimate of the total population of A. taiwanensis in the study area, but confirmed that Rincotrap^®, according to preliminary experiments [7,20], could be used in an integrated pest management perspective. On the other hand, a low percentage of the individuals caught in the control plot were found infected by B. bassiana. Thus, it is conceivable that mortality due to the treatment could be higher and due to other individuals present in the area, contaminated by spores dispersed into the environment, but not captured in the Rincotrap^® bands. The treatment with B. bassiana has the typical disadvantage of entomopathogenic fungi, i.e., a delay in efficacy, as highlighted in both semi-field and field trials. Indeed, considering the long average time elapsed between captures and deaths due to infection with B. bassiana, this would not exclude the possibility of reproductive events for contaminated adults of A. taiwanensis, as verified in the semi-field test.

5. Conclusions

The application of these combined techniques, Rincotrap^® bands treated with Naturalis^®, is promising, but multi-year experimentation, performed in diversified environments, will be necessary to better understand the potential field application of these integrated techniques. Furthermore, a monitoring system for the A. taiwanensis population and an evaluation of fig tree damage should be set up to assess this and other strategies to cope with this pest.