Morphophysiological Responses in Eucalyptus Demonstrate the Potential of the Entomopathogenic Fungus Beauveria bassiana to Promote Resistance against the Galling Wasp Leptocybe invasa

Abstract

The galling insect Leptocybe invasa Fisher & La Salle (Hymenoptera: Eulophidae) is a major limiting factor in the cultivation of drought-tolerant eucalyptus. The insect L. invasa is a major pest of eucalyptus plantations, and Beauveria bassiana (Bals.) Vuill (Ascomycota: Hypocreales) is being investigated as a potential biocontrol agent against this pest. The fungus B. bassiana can produce metabolites that affect insect biology and survival. Here, we investigated the ability of the entomopathogenic B. bassiana to endophytically develop and induce resistance to L. invasa in a drought-tolerant eucalyptus hybrid. In a greenhouse under semi-controlled conditions, a group of seedlings were sprayed with a solution containing the fungal spores of B. bassiana. The uninoculated seedlings and seedlings inoculated were infested with L. invasa, and their morphometric responses, gas exchange, and chlorophyll indexes were assessed. The number of leaves and height of the inoculated plants was higher than those of the uninoculated plants. The mean CO₂ assimilation rate (A) and transpiration rate (E) were higher for inoculated plants. The inoculated plants showed higher chl a and chl b contents. Compared to the uninoculated plants, the inoculated plants developed much fewer galls, while some showed only scar formations where L. invasa deposited its eggs. These results indicated that inoculating Eucalyptus with B. bassiana promoted resistance to L. invasa. To the best of our knowledge, this is the first study showing that an entomopathogenic fungus can develop endophytically to promote resistance against a galling insect pest.

1. Introduction

Planted forests play an important role in the global supply of forest services and products, thereby reducing the need to clear natural forests [1]. Eucalyptus L’Hér. is a popular choice for planted forests due to its fast growth, high productivity, adaptability, and raw materials for the pulp and paper industries [2]. The great genetic diversity of the genus allows us to select more adapted genotypes to adverse climates and have specific purposes, based on studies that evaluated the morphological and physiological potential of these plants and hybrids resulting from crossings [3].

Under predicted climate change, altered temperature and precipitation regimes are expected to increase the frequency and intensity of droughts in major eucalyptus-producing regions and inhibit the growth and sustainability of planted forests [4]. Indeed, drought events have become much worse due to greenhouse gas emissions, which are responsible for increasing the stresses caused by drought and heat, and favor climate-related pest outbreaks and fires [5]. This has inspired a renewed search for genotypes that are more tolerant to abiotic stresses, especially water stress. Among the Eucalyptus species with higher drought tolerance are E. camaldulensis, E. tereticornis, and hybrids of these species [6].

Insect pests are another major factor limiting the cultivation of eucalyptus. Some of these insects originated from Australia, the same place of origin of the Eucalyptus genus. When introduced to an exotic environment, these pests are presented with large amounts of food and few natural enemies, which promote their dissemination in eucalyptus plantations [7]. Leptocybe invasa Fisher & La Salle (Hymenoptera: Eulophidae) is a pest of Eucalyptus plantations around the world, especially to hybrids of E. camaldulensis and E. tereticornis. In attacked plants, this minute wasp from Australia induces galls along the entire length of the veins, petioles of young leaves, and the internodes of branch apices, which cause bending and atrophied growth in the attacked tissues and may lead the plant to death [8,9]. Galls modify the normal circulation of sap in vascular tissues, altering the production of metabolites associated with the photosynthetic process and affecting the development of infested plants, culminating in slower growth rates [10]. Climatic conditions and different eucalyptus host species affect the life cycle of L. invasa, since its best development occurs in warmer and drier conditions [11].

Plants do not have organs for locomotion, which makes it impossible for them to escape from herbivores and pathogens, making them dependent on other means to protect themselves. Plant defense can be constitutive, characterized by the development of structures that form a physical barrier to the foraging, feeding, and oviposition of insects, or by the production of chemical substances which can act as toxins for herbivores, which have always been present in the plant [12]. The other form of plant defense is induced, which consists of defense-related structures and metabolites that are usually present at low levels but become abundant after detection of the presence of pathogens or herbivores. In this way, the induced defense remains disabled until the attack is detected, avoiding metabolic costs [13]. Despite all these defense mechanisms that plants have acquired over different evolutionary processes, insects have also developed evolutionary adaptations that allow them to avoid or overcome these mechanisms [14].

The use of endophytic organisms can be an effective alternative to control pest insects, since the plant–microorganism association can strengthen the plant defenses, reduce damage caused by pathogens or insects, accelerate seed germination, promote plant equilibrium in adverse conditions, and increase plant growth [15]. These naturally occurring organisms comprise a diverse group of entomopathogens that can control pest insect populations [16]. The success of endophytic colonization depends on factors, such as plant age, inoculation method, fungal species, direct exposure to sunlight, and rain. Inoculation can be performed in a variety of ways, such as soil soaking, foliar spraying, stem injection, irrigation, or seed treatment [17].

The fungus Beauveria bassiana (Bals.) Vuill (Ascomycota: Hypocreales) is a commonly used biocontrol agent in agricultural crops and is considered a viable alternative to synthetic insecticides. When used in conjunction with other biocontrol organisms, such as nematodes and parasites, and good agricultural practices, B. bassiana has been shown to reduce the use of chemical pesticides and can be an important tool within an integrated pest management system [18]. It has also been shown that B. bassiana may produce metabolites that directly or indirectly affect insects through antibiosis or antixenosis [19].

Playing a double biological role, the fungus B. bassiana can act both as an endophytic and entomopathogen. In both life stages, the fungus secretes a wide range of extracellular enzymes, such as chitinases, proteases, lipases, amylases, and lacases. This variety of extracellular enzymes makes this organism a potential source of industrial enzymes and other important biomolecules [20]. B. bassiana has the ability to colonize host tissues above and below ground [21]. In the host, this association promotes an increase in growth and biomass, in addition to providing protection against pest insects and phytopathogens through the production of secondary metabolites that induce systemic resistance [22].

The use of B. bassiana as an endophytic fungus is an economically viable alternative to more expensive methods that use formulations containing fungal propagules and synergists. B. bassiana is also protected inside the plant against abiotic factors and can therefore persist in the crop population [23]. In this study, we hypothesized that the use of the entomopathogenic fungus B. bassiana, as a potential resistance-inducing agent, will promote resistance of the E. tereticornis × E. camaldulensis hybrid to L. invasa attacks. Accordingly, we measured the morphometric responses, gas exchange, and chlorophyll indexes of Eucalyptus infested with L. invasa after inoculation with B. bassiana and compared with uninoculated plants

2. Materials and Methods

2.1. Plant Material and Breeding of L. invasa

The seedlings used in this study came from a clonal matrix of E. tereticornis Sm × E. camaldulensis Dehnh (VS058), susceptible to L. invasa, produced in the nursery of the Laboratory of Plant Health—Applied and Functional Ecology, Federal University of Tocantins (Gurupi, Brazil). The seedlings used were produced in plastic tubes with a capacity of 55 cm³, containing commercial substrate Bioplant^® (Bioplant, Ponte Nova, MG, Brazil), arranged in trays with support for ninety-six tubes. The fertilization of the seedling production phase was carried out using 5 g of NPK (10; 10; 10) and 3 g of FTE BR 12 (sulfur: 3.9%; boron: 1.8%; copper: 0.85%; manganese: 2.0%; and zinc: 9.0%). Irrigation was conducted by sprinkling, performed four times a day, lasting five minutes each. The seedlings produced were kept in a greenhouse until the experiment was carried out. At 120 d of age, the seedlings were transplanted into 3.6 L pots containing Bioplant^® commercial substrate and acclimated in a greenhouse for 30 d before inoculation with the fungus.

The rearing of L. invasa started with seedlings of clone VS058 donated by Braxcel Florestal S.A. being parasitized by wasps, which were placed in wooden rearing cages covered with organza fabric (2.5 m wide; 2.6 m long; 3.0 m high). Wasp specimens were identified by Prof. Carlos Frederico Wilcken of the Department of Plant Production of the São Paulo State University—Júlio de Mesquita Filho (FCA-UNESP). Healthy seedlings were continually added to the cage for egg laying.

2.2. Experimental Design

The experiment was conducted in a greenhouse at the experimental research station of the Federal University of Tocantins (UFT), at the University Campus of Gurupi (11°43′ S and 49°04′ W, 284 m altitude). The experimental design was entirely randomized, and the plants of both treatments were randomly placed in the experiment site to guarantee unbiased results. The experiment had two treatments comprised of eight seedlings each: uninoculated seedlings and seedlings inoculated with B. bassiana (both treatments were infested with L. invasa).

The fungus B bassiana (Bals.) Vuill (Beauveril strain PL 63) was procured from the mycological collection of the Laboratory of Insect–Microorganism Symbiosis at the UFT/Gurupi Campus. To produce the conidia suspension, the isolates were plated on 50 mm Petri dishes containing PDA (Potato Dextrose Agar) medium plus 500 mg amoxicillin antibiotic and incubated in a BOD (Biochemical Oxygen Demand) chamber at 25 ± 2 °C and a 12 h photoperiod for 7 to 10 d.

The spores from the colonies were removed under aseptic conditions in a flow chamber. The plates containing the colonies were treated with 10 mL of autoclaved distilled water with 0.002% (v/v) Tween 80^® adhesive spreader, followed by delicate scraping with a sterile spatula to collect and transfer spores to a sterile becker containing 100 mL of sterile distilled water. The solution with mycelium-containing spores was stirred for 10 min in an incubator with orbital shaking at 150 rpm and room temperature. The suspension was then filtered through double-layered sterile gauze to retain the mycelium fragments and the remains of the culture medium, and then this suspension was transferred to an autoclaved becker. An aliquot of the filtered spore suspension was placed in a Neubauer chamber to count the spores/mL using an optical microscope. The inoculum was adjusted to a concentration of 10⁸ spores/mL.

After being transplanted into pots, the plants underwent an acclimatization period, where they remained in a greenhouse for 30 days. After the acclimatization period, the Eucalyptus seedlings were sprayed with the inoculum containing the B. bassiana spores. To facilitate the entry of the spores, lesions were made by rubbing the rough end of a kitchen sponge on the surface of the fourth, fifth, and sixth fully expanded leaf of each seedling before inoculation. After inoculation, the plants were covered with sterile transparent plastic bags for a period of 48 h in order to maintain a high level of humidity after inoculation.

Eucalyptus seedlings were infested with L. invasa 45 days after inoculation. Each seedling had a minimum number of six apices wrapped in an organza bag containing a microtube with two L. invasa females. The wasps were collected in the rearing cage of the Plant Health Laboratory—Applied and Functional Ecology of the Federal University of Tocantins (Gurupi, Brazil). The microtubes were opened and infestation by the wasps was conducted for 48 h, after which the wasps were removed along with the bags.

During the experiment, the plants were fertilized fortnightly and interspersed with 5 g of NPK (10; 10; 10) and 3 g of FTE BR 12.

2.3. Morphophysiological Analysis of Eucalyptus Plants

Morphometric evaluations were performed at 15, 30, 45, 65, 80, 95, and 110 days after inoculation (d.a.i.) of the seedlings with B. bassiana. The following parameters were measured: plant height, stem diameter, number of fully expanded leaves, and number of twigs. Plant height was obtained in centimeters, from the base of the plant to the apex of the aerial part, with the aid of a millimeter ruler. Stem diameter was measured in millimeters at the base of the plants with a digital caliper. For each plant, at each evaluation time, the number of fully expanded leaves and the number of twigs were counted. At the end of the experiment, the increment between each evaluation time was calculated for each evaluated parameter.

Physiological analyses were performed after 1, 15, 30, 50, 65, 80, 95, and 110 d.a.i. The measured parameters included gas exchange (net CO₂ assimilation, transpiration, intercellular carbon, and stomatal conductance) and the chlorophyll index (chl a and chl b). Gas exchange was measured using an LCi-SD gas exchange meter (ADC Bioscientific, Hoddesdon, England). The chlorophyll index was obtained using an electronic chlorophyll meter chlorofiLOG^® (Falker, Porto Alegre, Brazil).

2.4. Evaluation of Gall Development

The number of galls on each seedling was counted 31 days after infestation with L. invasa, on the 80th d.a.i. We simultaneously recorded a morphological description of the galls found in both groups.

2.5. Statistical Analysis

A Shapiro–Wilk test was performed in order to verify the normality of the data. A Levine test was also performed to verify the homogeneity of variance. Because the data followed a normal distribution and the variances were determined to be homogeneous, the means of each group were compared using Student’s t-test, with statistical significance set at p < 0.05. All analyses were performed using Sisvar 5.6 software [24]. Data are expressed as the mean ± standard error.

3. Results

3.1. Differential Growth of Eucalyptus after Inoculation with B. bassiana

On the 30th d.a.i., the uninoculated plants were shorter than the inoculated and infested plants (p = 0.015). We also observed that after infestation with L. invasa, the plants inoculated with B. bassiana did not suffer an abrupt reduction in height compared to the uninoculated plants, especially at 95 d.a.i. (p < 0.01), where a reduction of 50.35% was observed relative to day 80 (Figure 1A). Except on the 65th d.a.i., where the uninoculated plants showed a greater increase in diameter (p = 0.045), no significant differences were identified on the other days (Figure 1B).

Regarding the number of twigs, a significant difference was found on the 30th d.a.i., where uninoculated plants had more twigs than the inoculated plants (p = 0.001). No other significant differences were identified between the treatment groups on the other evaluation days (Figure 1C). Regarding leaf number, inoculated plants at 15 d.a.i. had more leaves than uninoculated plants (p = 0.003). However, uninoculated plants at 30 d.a.i. presented a higher mean number of leaves compared to those of the inoculated plants (p = 0.035). Regarding the other parameters evaluated, no significant differences were found between the treatments (Figure 1D).

3.2. Gas Exchange Is Affected after Inoculation with B. bassiana in Eucalyptus

Regarding the CO₂ assimilation rate, we observed a significant difference on 30, 50, 65, and 80 d.a.i. (p < 0.001, p = 0.0034, p = 0.0011, and p = 0.0074, respectively), and only on the 30th d.a.i. did the uninoculated plants show the highest mean. On the remaining days, the assimilation rate was normal and even increasing, while the uninoculated plants suffered an abrupt drop in CO₂ assimilation rate after infestation with L. invasa. No other significant differences were identified between the treatment groups on the other evaluation days (Figure 2A). Transpiration was significantly higher in inoculated plants on 30, 80, and 95 d.a.i. compared to that in the uninoculated plants (p = 0.0001, p = 0.0187, and p = 0.0371, respectively). On the other days evaluated, no significant differences were found between the treatment groups (Figure 2B).

Intercellular carbon levels differed between treatment groups on 30 and 50 d.a.i. On the 30th d.a.i., the inoculated plants presented the highest mean (p = 0.0063), while the uninoculated plants presented the highest mean on the 50th d.a.i. (p = 0.0323). No other significant differences between the treatment groups were identified on the other evaluation days (Figure 2C). Mean stomatal conductance only differed between groups at 15 d.a.i., with uninoculated plants having the higher mean (p = 0.0406). For the other evaluation days there was no significant difference (Figure 2D).

3.3. Chlorophyll Content in Eucalyptus in Response to Inoculation with B. bassiana

On 15, 30, 65, and 95 d.a.i., the uninoculated plants showed the highest mean chl a index compared to that of the inoculated plants (p = 0.010, p = 0.002, p = 0.0003, and p < 0.001, respectively). No significant difference was detected on the other days of evaluation (Figure 3A). The same was observed for the chl b index, where the uninoculated plants showed the highest averages on 30 and 95 d.a.i. (p < 0.001 and p = 0.001, respectively). No significant difference was found on the other days of evaluation (Figure 3B).

3.4. Assessment of Gall Development

We characterized and counted the number of galls per plant at 31 d after infestation with L. invasa. Inoculated plants showed practically no galls, with an average of less than one gall per plant (Figure 4A). The uninoculated plants showed the formation of galls on the veins, petioles of the leaves, and internodes of the apices of the branches (Figure 4B). Unlike in the uninoculated plants that developed galls, some inoculated plants showed the formation of a scar in the veins that L. invasa laid its eggs in (Figure 4C), though no other symptoms characteristic of insect infestation were observed.

4. Discussion

The present study proposes the use of the entomopathogenic microorganism B. bassiana as an alternative biological agent for the control of the galling insect L. invasa, which poses a serious global problem in eucalyptus cultivation. Although B. bassiana is already used commercially in agricultural crops [18], little is known about how and to what extent it impacts the development of L. invasa. Our results showed that inoculation of eucalyptus seedlings with B. bassiana promoted plant resistance to L. invasa. The inoculated plants experienced delayed growth on the first days after inoculation, before the infestation, especially in height and number of twigs. However, from 65 d.a.i. onwards, no differences in plant growth were observed between the inoculated and uninoculated plants. Similar results were observed in grapevines by Mantzoukas et al. [25]. The effects of inoculation with B. bassiana were positive in corn [26], resulting in increased growth under greenhouse conditions. Similar positive effects were observed in our study, especially in the first 15 d.a.i. in terms of leaf number and 95 d.a.i. in terms of height. This increase in growth was likely associated with the increase in CO₂ assimilation rate [27] observed in the inoculated plants.

Endophytic fungi tend to have neutral effects on plant physiology [28], as observed in sorghum plants, where the presence of B. bassiana did not interfere with gas exchange [29]. However, we observed adverse effects of inoculation on gas exchange: in the first 30 d.a.i., the plants showed the lowest rates of CO₂ assimilation and stomatal conductance, and the highest transpiration and intercellular carbon concentrations. Higher intercellular carbon concentrations suggest leaf biochemical limitations, such as in Rubisco activity [30]. This increase in intercellular carbon concentrations may have been caused by the inoculation, and further research should investigate the biochemical reactions associated with this increase. After 2 d of infestation with L. invasa, we observed a decrease in the CO₂ assimilation rate for uninoculated plants, while the CO₂ assimilation rates in inoculated plants were relatively normal and even increased gradually until 80 d.a.i. These results correspond with the changes in height, with greater CO₂ assimilation supporting greater production of photoassimilates for plant growth [31].

Another adverse effect of inoculation also observed within the first 30 d was higher mean chl a and chl b indexes. Higher mean chl a and chl b indexes were also found after infestation with L. invasa. In addition to chlorophyll being responsible for regulating the absorbed solar radiation, leaf chlorophyll concentrations have a strong positive relationship with photosynthetic and carbon fixation rates [32]. This may be related to the redistribution of abundant photosynthates to promote overall growth. Increases in the chl a and b indexes were also observed in E. pellita seedlings inoculated with the fungus Amanita pantherina [33].

Although we observed that there was oviposition of the insect L. invasa on the inoculated plants, after 30 d of infestation, the inoculated plants showed few to no galls, while the uninoculated plants showed normal gall development. Acting as an endophytic when present inside the plants, the fungus B. bassiana demonstrated the ability to produce compounds capable of defending against pest attacks [19,20]; in light of this, it seems reasonable to infer that B. bassiana reduced and even inhibited the development of galls, producing compounds that prevented the development of the insect inside the plant. The formation of galls leads to rapid changes in the size of the leaves and the galls, due to the hyperplasia (multiplication exaggerated of cells), and metaplasia (transformation of normal tissue into gall tissue), caused by the insect attack. During this process, plants undergo rapid and excessive cell growth, and a change from normal to gall tissue, all of which results in folding and stunting of the attacked tissues, consequently inhibiting growth [8,9]. This explains why the uninoculated plants showed less growth than the inoculated plants.

The formation of galls by L. invasa strongly interferes with the hydraulic architecture of E. camaldulensis, including the density of minor veins and stomata. In addition, chlorophyll contents, gas exchange, and photosynthetic electron flow were reduced in infested plants [34]. Given that the potential effects of inoculation on hydraulic architecture and gas exchange were null, it is reasonable to infer that the uninoculated plants had lower transpiration values after infestation. This is because transpiration in plants with galls is severely affected by the reduction in the number of leaves and total leaf area [34]. However, there may be effects of inoculation that were not elucidated in this study, and further studies are needed to investigate the influence of B. bassiana on hydraulic architecture, minor vein density, and stomatal density.

5. Conclusions

Our findings showed that eucalyptus seedling inoculation with B. bassiana prevented growth impairment after infestation by L. invasa. Morphophysiological adaptations, such as increased CO₂ assimilation rates and consequently increased production of photoassimilates, together with increased chlorophyll a and b contents, culminated in increased growth of the inoculated plants. Notably, the development of galls was limited or nonexistent on the inoculated plants.

Collectively, our results indicated that inoculation with the fungus B. bassiana adequately promoted resistance in eucalyptus against L. invasa. Considering the complexity of this tri-trophic interaction between eucalyptus, the pest L. invasa, and fungus B. bassiana, more comprehensive studies are needed to evaluate changes in anatomy, hydraulic architecture, primary and secondary metabolism, and the expression of related genes. These studies will be of utmost importance to improving the management of eucalyptus under predicted climate change.