*Article* **The Arbuscular Mycorrhizal Fungus** *Glomus viscosum* **Improves the Tolerance to Verticillium Wilt in Artichoke by Modulating the Antioxidant Defense Systems**

**Alessandra Villani , Franca Tommasi and Costantino Paciolla \***

Department of Biology, University of Bari "Aldo Moro", Via E. Orabona 4, 70125 Bari, Italy; alessandra.villani@uniba.it (A.V.); franca.tommasi@uniba.it (F.T.) **\*** Correspondence: costantino.paciolla@uniba.it; Tel.: +39-080-544-3557

**Abstract:** Verticillium wilt, caused by the fungal pathogen *Verticillium dahliae*, is the most severe disease that threatens artichoke (*Cynara scolymus* L.) plants. Arbuscular mycorrhizal fungi (AMF) may represent a useful biological control strategy against this pathogen attack, replacing chemical compounds that, up to now, have been not very effective. In this study, we evaluated the effect of the AMF *Glomus viscosum* Nicolson in enhancing the plant tolerance towards the pathogen *V. dahliae*. The role of the ascorbate-glutathione (ASC-GSH) cycle and other antioxidant systems involved in the complex network of the pathogen-fungi-plant interaction have been investigated. The results obtained showed that the AMF *G. viscosum* is able to enhance the defense antioxidant systems in artichoke plants affected by *V. dahliae*, alleviating the oxidative stress symptoms. AMF-inoculated plants exhibited significant increases in ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), and superoxide dismutase (SOD) activities, a higher content of ascorbate (ASC) and glutathione (GSH), and a decrease in the levels of lipid peroxidation and hydrogen peroxide (H2O2). Hence, *G. viscosum* may represent an effective strategy for mitigating *V. dahliae* pathogenicity in artichokes, enhancing the plant defense systems, and improving the nutritional values and benefit to human health.

**Keywords:** Verticillium wilt; *Glomus viscosum* Nicolson; arbuscular mycorrhizal fungi; oxidative stress; antioxidant systems; defense ability

### **1. Introduction**

The artichoke (*Cynara scolymus* L.) is a horticultural species of relevant economic interest belonging to the Asteraceae family, widely cultivated in the Mediterranean basin and widespread throughout the world [1,2]. This perennial crop is well known for the antioxidative, antimicrobial, and probiotic properties of its edible parts, including the inner fleshy leaves (bracts) and the receptacle [1,3]. Several studies have demonstrated that even some non-food by-products of artichokes, including leaves, external bracts, and stems, exhibit beneficial and therapeutic effects and are widely used as hepatoprotective [4], antioxidant [5,6], anticarcinogenic [7], hypoglycemic [8], and hypocholesterolemic [9] agents. The health-promoting properties and important nutritional values of artichokes have been extensively related to inulin, fibers, and minerals, and to the high content of some bioactive phenolic compounds, such as caffeoylquinic acid derivatives and flavonoids, showing a strong scavenging activity against reactive oxygen species (ROS) and free radicals [1,2,10].

As the artichoke is an herbaceous plant which survives in the field for several years, a large number of insects, nematodes, bacteria, fungi, and viruses can attack and invade its seeds, roots, foliage, and vascular system, causing numerous diseases [11,12]. Verticillium wilt, caused by the fungus *V. dahliae* Kleb., represents one of the greatest threats to the artichoke plantation worldwide [13–15]. This soil-borne pathogen is distributed throughout

**Citation:** Villani, A.; Tommasi, F.; Paciolla, C. The Arbuscular Mycorrhizal Fungus *Glomus viscosum* Improves the Tolerance to Verticillium Wilt in Artichoke by Modulating the Antioxidant Defense Systems. *Cells* **2021**, *10*, 1944. https:// doi.org/10.3390/cells10081944

Academic Editor: Suleyman Allakhverdiev

Received: 24 June 2021 Accepted: 28 July 2021 Published: 30 July 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

the world, and it affects over 400 plant species that belong to 14 plant families, showing a broad range of symptoms and causing significant yield losses and quality reduction for most of the host plant species [15,16]. Among these species, almond, apricot, artichoke, cabbage, cauliflower, chrysanthemum, cotton, cucurbits, olive, peach, potato, strawberry, sunflower, and tomato were defined as the most severely affected host crops [16]. *V. dahliae* causes a monocyclic disease divided into three phases: dormant, parasitic, and saprophytic [17,18]. In the presence of a host, the disease cycle begins with germination of microsclerotia that are released in the soil with the decomposition of plant materials where they can remain viable for up to 14 years. Hyphae from germinating microsclerotia can colonize and penetrate the roots of host plants, following a slow progression through the vascular (xylem) tissues, where conidia can grow and continue the colonization, leading to xylem malfunctioning and reduced movement of water and nutrients from the roots to the foliage of the infected host [17]. Over the years, several strategies have been tested to manage diseases caused by *V. dahliae*, including selection of planting site, crop rotation and manipulation of fertility and irrigation, use of healthy planting material, selection of available resistant cultivars, fungicides treatments, and soil fumigants [15,18–21].

Methyl bromide has been widely used for decades as a soil fumigant for controlling Verticillium wilt until its complete phase-out in 2005 according to the Regulation EC 2037/2000 because of its threat to the environment, being one of the major ozone depleting substances, and to humans, causing lung injury and neurological effects. Some other fumigants have been tested, such as the mixture of 1,3-dichloropropene and chloropicrin, dazomet, and metam sodium, but are not very effective [22]. Similarly, several fungicides applied as foliar sprays, soil drenches, or granular preparations have been tested, but the effectiveness was observed only with high dosages, which many a times cause phytotoxic effects [23]. Therefore, the inability of fungicide and soil fumigants treatments to control the disease successfully, the unavailability of artichoke resistant cultivars as well as the inaccessibility of *V. dahliae* during infection and its long-term persistence in the field have required alternative strategies. Furthermore, the public concern over the environment pollution, ecosystem's biodiversity, and food safety has enhanced research efforts towards eco-friendly practices for a sustainable agricultural management. To ensure that aim, beneficial microorganisms, such as AMF, could play a crucial role. AMF are symbionts, mainly belonging to the phylum Glomeromycota that form arbuscular mycorrhizal associations with the roots of over 80% of all vascular plants [24]. Numerous studies have demonstrated that AMF can improve water and mineral nutrient uptake from the soil by increasing the plant root surface area [25]. Initial stages of AMF colonization trigger an intracellular ROS burst in the host plant; however, this effect is transient and is overcome by enhanced activities of antioxidant enzymes [26]. Indeed, AMF increase the accumulation of secondary metabolites in several plants, including phenolic compounds, vitamins, and sugars [10,27], mitigate the oxidative burst of plants under abiotic stresses by increasing the activity of some antioxidants that are involved in the alleviation of oxidative damage caused by ROS [28–30], and protect host plants from pathogens, overcoming the harmful effects of abiotic and biotic stresses [31]. In particular, the efficacy of AMF in reducing the disease severity of Verticillium wilt has been demonstrated on olive [32,33], eggplant and tomato [34,35], pepper [35], oilseed rape (*Brassica napus* L. cv. Licosmos), and strawberry (*Fragaria ananassa* cv. Elsanta) [36] plants. Moreover, the presence of an autochthonous mycorrhizal consortium "Rhizolive consortium" on the early oxidative events induced in olive plants after *V. dahliae* inoculation stimulated the activity of antioxidant enzymes, reducing oxidative damage [37], and treatments with six AMF in two artichoke cultivars increased the level of total phenols and total antioxidant activity [20].

The increase in ROS is a common biochemical response to abiotic and biotic stresses in plants. Higher ROS levels in the cell could cause oxidative damage to DNA, lipids, and proteins. It is well known that the ROS level in cells is under the control of antioxidant systems, such as the ASC-GSH cycle [38], and enzymes, including SOD, catalase (CAT), and generic peroxidases (PODs), which have a pivotal role in defense mechanisms. The

activity of those enzymes, with that of APX, a component of ASC-GSH cycle, is crucial for determining the steady-state level of superoxide anion and H2O2 in plant cells [39]. SOD dismutates superoxide anion to H2O2, which can be converted into oxygen and H2O by CAT, PODs, or APX. In the ASC-GSH cycle, the APX uses two molecules of ASC to reduce H2O2 to water, with the concomitant generation of two molecules of monodehydroascorbate (MDHA) [40]. MDHA is a radical with a short lifetime that is rapidly reduced to ASC by MDHAR, which is a flavin enzyme that utilizes NAD(P)H as electron donors. Dehydroascorbate (DHA), the oxidized form of ASC, can be reduced back to ASC by DHA reductase (DHAR), which utilizes GSH as an electron donor, leading to the formation of glutathione disulphide (GSSG), which is in turn re-reduced to GSH by NADPH, a reaction catalyzed by the glutathione reductase (GR).

The objective of this study was to test the ability of the AMF *G. viscosum* to moderate the metabolic alterations related to oxidative stress in artichoke plants attacked by *V. dahliae*. In particular, a deeper investigation of the involvement of the defense systems in the fungiplant interaction, by evaluating the level of the components of the ASC-GSH cycle and the antioxidant enzymes (CAT and SOD), led to a better understanding of the biochemical mechanism on the basis of this complex network among the plant, pathogen, and AMF.

### **2. Materials and Methods**

### *2.1. Chemicals*

All reagents used in this study were of the highest grade available, purchased from Sigma–Aldrich (Milan, Italy) and used without further purification. Ultrapure water was produced by a Milli-Q system 84 (Millipore, Bedford, MA, USA).

### *2.2. Plant Material and Sampling*

The material analyzed was obtained from the experimental farm of "P. Martucci" of the University of Bari in Valenzano, Apulia, Italy. Plants of artichokes (*Cynara cardunculus* L. var. *scolymus* L. cv. Violetto di Provenza), obtained by micropropagation, were transplanted in pots containing a commercial peat mixture soil enriched with nutrients (organic carbon 46%, organic nitrogen 1–2%, organic matter 80%) and mixed with perlite at a 2:1 (*v*/*v*) ratio. The peat mixture was sterilized and used to fill 10 cm diameter pots. Prior to transplantation, half of the microplants were inoculated with 10 g of crude inoculum of the AMF *G. viscosum*, as described by Morone Fortunato et al. [41]. Non-mycorrhizal plants were used as controls. Acclimatization took place in greenhouse conditions at 18 ± 2 ◦C with mist and a relative humidity level reduced from 85–90% to 55–60% over 20 days. After 60 days, 48 non-mycorrhizal plants and 48 inoculated plants were transplanted to the open field, according to a randomized block design with treatments replicated three times. Each block consisted of eight plants, and the spacing used was 1.2 m between rows and 1 m between plants for all the treatments. A portion of the field was inoculated 15 days before the transplantation with an inert substrate enriched with mycelium, microsclerotia, and spores of *V. dahliae* isolated from naturally infected artichokes. Then, the treatments were established as follows: (i) non-mycorrhizal plants (Ctrl), (ii) non-mycorrhizal plants inoculated with *V. dahliae* (I), (iii) mycorrhizal plants (M), (iv) mycorrhizal plants inoculated with pathogen (MI).

### *2.3. Source of V. dahliae Isolates*

Isolates of *V. dahliae* were recovered from symptomatic artichoke plants in a field with a known history of Verticillium wilt. Segments of 5 mm long surface-sterilized stems from infected host plants were transferred on a potato dextrose agar (PDA), (Difco, Detroit, MI, USA) supplemented with streptomycin sulphate (100 ppm) and incubated at 27 ◦C for 10 days in darkness. Colonies of *V. dahliae* were morphologically identified visually and microscopically, subcultured on PDA without antibiotics, and then mixed with vermiculite for the inoculation in the field. A further identity confirmation was provided by sequencing. PCR amplification and sequencing were performed using the ribosomal in-

ternal transcribed spacer region (*ITS*) as the locus, according to Inderbitzin et al. 2011 [42]. Species identification was confirmed by BLASTn against the NCBI GenBank database (http://www.ncbi.nlm.nih.gov, accessed on 14 January 2021).

### *2.4. Disease Assessments*

A scoring metric was used to assess disease severity of the artichoke plants over time. Wilt severity was rated according to Uppal et al.'s [43] scoring system as follows: 0, no wilt symptoms; 1, inter-veinal chlorosis on the lower leaves; 2, moderate necrosis and defoliation of the lower leaves; 3, severe leaf necrosis and defoliation; and 4, severe defoliation accompanied by pronounced stunting, chlorosis, and necrosis of the remaining leaves. Furthermore, a rating scale was also established to evaluate the severity of vascular browning of artichoke stems. This scale consisted of the following grades: 0, no vascular browning; 1, trace to less than 9% of the stem cross-section showing a vascular browning; 2, 10–24% of the stem cross-section with a vascular browning; 3, 25–49% of the stem crosssection showing vascular browning; and 4, over 50% of the stem cross-section exhibiting vascular browning. In addition, the effect of mycorrhization on the growth of artichoke plants was assessed by counting the number of flower heads per plant. Data were analyzed with analysis of variance (ANOVA), and the means were compared by the Duncan test.

### *2.5. Determination of ASC and GSH Pool Contents*

Foliar tissues (20 g) were homogenated at 4 ◦C with three volumes of 5% (*w/v*) metaphosphoric acid and then centrifuged for 15 min at 20,000× *g*. The resulting supernatant was used for analysis of the ASC and GSH pool content according to Zhang and Kirkham [44].

### *2.6. Enzyme Assays*

For determination of antioxidant enzyme activities, samples were homogenized according to Mastropasqua et al. [45] with slight modifications. Briefly, twenty grams of foliar tissues were homogenized in 50 mM Tris–HCl, pH 7.8 containing 0.3 mM mannitol, 1 mM EDTA, 10 mM MgCl2, 1% (*w/v*) polyvinyl-pyrrolidone (PVP), and 0.05% (*w/v*) cysteine 1%, at 4 ◦C. The homogenate was filtered through four layers of cheesecloth and centrifuged (20,000× *g*, 20 min, 4 ◦C). The supernatant was desalted by dialysis against 50 mM Tris-HCl, pH 7.8, and used for spectrophotometric analysis of the total proteins and enzymatic activities.

The total protein content of samples was measured with a Protein Assay kit from Bio-Rad (Hercules, CA, USA) with bovine serum albumin as the standard [46]. The reproducibility of the Bio-Rad kit, expressed as the coefficient of variation (%CV), is 2% approximately; the lower limit of the detection for protein molecular weight is 3000 to 5000 Daltons.

The enzymatic spectrophotometric assays for the determination of cytosolic APX (EC 1.11.1.11), DHAR (EC 1.8.5.1), CAT (EC 1.11.1.6), GR (EC 1.8.1.7), MDHAR (EC 1.6.5.4), and SOD (EC 1.15.11) were performed according to Paciolla et al. [47] and Mastropasqua et al. [48].

### *2.7. Electrophoretic Analyses*

Native-Polyacrilamide Gel Electrophoresis (Native-PAGE) was performed on PAGE (4.3% T; 7.3% C) with a running buffer composed of 4 mM Tris-HCl pH 8.3 and 38 mM glycine. In each lane of the gel, 200 μg of total proteins were loaded. After the electrophoretic run, the gels were washed with distilled water and incubated in specific buffers for the detection of APX and CAT, as described in Paciolla et al. [49]. For the SOD, the activity on the gel was visualized by incubating it in 0.053 Tris–HCl buffer pH 8.2 containing 0.21 mM riboflavin and 0.244 mM nitro-blue tetrazolium (NBT) in the dark; after 15 min, achromatic bands on a grey background appeared, a 50% glycerol solution was used to block the reaction.

For densitometric analysis of SOD activity, the gel was acquired utilizing the Gel/ChemiDoc and Quantity One software (BioRad Laboratories Inc., Milan, Italy) to obtain information on the changes in the activity of each band due to different treatments. A relative value of 100 was assigned to the intensity of the bands of Ctrl and I samples.

### *2.8. Lipid Peroxidation Analysis and H2O2 Content*

For lipid peroxidation, plant material was ground with four volumes of 0.1% (*w/v*) trichloroacetic acid (TCA). The homogenate was centrifuged at 12,000 × *g*, for 10 min, at 4 ◦C. One mL of the supernatant was mixed with 4 mL of 20% TCA containing 0.5% (*w/v*) thiobarbituric acid (TBA). The level of cell lipid peroxidation was evaluated in terms of malondialdehyde (MDA) content determined by the TBA reaction as described by Zhang and Kirkham [44]. Intracellular H2O2 concentration was evaluated according to Lee and Lee [50].

### *2.9. Statistical Analysis*

The biochemical data presented are the means of five different experiments. Statistical analysis was done using Student's *t*-test, with level of significance for *p* < 0.05 and highly significant for *p* < 0.01; the standard deviation (SD) was calculated, and its range is shown in the figures. Data presented for disease assessments are the average of three experiments with three replicates and were analyzed with ANOVA with *p* ≤ 0.05; the means were compared by the Duncan test.

### **3. Results**

### *3.1. Disease Assessments*

The results showed a beneficial effect of mycorrhization in containing artichoke wilt. In particular, the AMF *G. viscosum* significantly reduced the disease severity, measured by symptoms' development on leaves, on the MI treatment, while it slightly reduced the vascular browning (Table 1). In addition, the beneficial effect of mycorrhization on productivity was observed.

**Table 1.** Effectiveness of mycorrhizal fungus *G. viscosum* in protecting globe artichoke against Verticillium wilt. For each parameter, different letters (a,b,c) within the same column indicate that the means are significantly different at *p* ≤ 0.05 according to Duncan test. The experiments were repeated three times with three replicates. Ctrl, non-mycorrhizal plants; I, non-mycorrhizal plants inoculated with *V. dahliae*; M, mycorrhizal plants; MI, mycorrhizal plants inoculated with *V. dahlia*.


### *3.2. Ascorbate and Glutathione Pool Content*

The mycorrhizal plants inoculated with the pathogen *V. dahliae* (MI) showed an increase (*p* < 0.05) in ASC content as compared to non-mycorrhizal inoculated plants (I), while no significant increment was observed in mycorrhizal plants (M) with respect to non-mycorrhizal plants (Ctrl) (Figure 1, Panel a). DHA did not differ significantly among the treatments (data not shown), and, hence, the ascorbate redox ratio (ASC/ASC + DHA) was higher in MI than in the other treatments (Figure 1, Panel b).

**Figure 1.** Ascorbate (ASC) content (**a**) and ASC redox ratio (**b**) in artichoke control plants (Ctrl), in mycorrhizal plants (M), in plants inoculated with *Verticillium dahliae* (I), and in mycorrhizal plants inoculated with *V. dahliae* (MI). The results are given as the mean values of at least five experiments ± SD; indicates values significantly different from the artichoke inoculated with *V. dahliae* (I) by the Student's *t* test with *p* < 0.05. FW, fresh weight.

In both mycorrhizal plants (M and MI), an increase (*p* < 0.05) in GSH content was observed (Figure 2, Panel a) with respect to the control and the inoculated plants (I), respectively. In addition, the GSSG content was similar in all samples (data not shown), therefore, the glutathione redox ratio (GSH/GSH + GSSG) was higher in M and MI with respect to the control and the plants inoculated with the pathogen (I), respectively (Figure 2, Panel b).

**Figure 2. Glutathione** (GSH) content (**a**) and glutathione redox ratio (**b**) in artichoke control plants (Ctrl), in mycorrhizal plants (M), in plants inoculated with *Verticillium dahliae* (I), and in mycorrhizal plants inoculated with *V. dahliae* (MI). The results are given as the mean values of at least five experiments ± SD; \* indicates values significantly different from the control (Ctrl) by the Student's *t* test with *p* < 0.05; indicates values significantly different from the artichoke inoculated with *V. dahliae* (I) by the Student's *t* test with *p* < 0.05. FW, fresh weight.

### *3.3. Antioxidant Enzyme Assays*

Activities of the enzymes in the ascorbate–glutathione cycle, including APX, DHAR, MDHAR, and GR, showed difference trends. The activity of APX was significantly higher in MI compared to I as shown in Figure 3 (Panel a). Similarly, M showed higher APX activity with respect to the control. These results were confirmed by Native-PAGE, which showed that enzyme activity of APX was higher in M and MI than in Ctrl and I, respectively.

Furthermore, the native electrophoretic pattern of APX showed a total number of three isoforms with same migration rate in all samples (Figure 3, Panel b).

**Figure 3.** Spectrophotometric activity (**a**) and electrophoretic profile after Native-PAGE (**b**) of ascorbate peroxidase (APX) in the cytosolic fraction of artichoke control plants (Ctrl), mycorrhizal plants (M), plants inoculated with *Verticillium dahliae* (I), and in mycorrhizal plants inoculated with *V. dahliae* (MI); 1 U = 1 nmol of ascorbate oxidized min<sup>−</sup>1; prot. = proteins. The results are given as the mean values of at least five experiments ± SD; \* \* and - indicate values significantly different from the control (Ctrl) and from the artichoke inoculated with *V. dahliae*, respectively, by the Student's *t* test with *p* < 0.01.

The assay of GR and MDHAR activities revealed a trend similar to APX, showing both in M and MI a significant increment compared to Ctrl and I plants, respectively (Figure 4, Panels a and b). In contrast, the activity of DHAR remained almost unchanged among all treatments (data not shown).

**Figure 4.** Enzymatic activity of glutathione reductase (GR) (**a**) and monodehydroascorbate reductase (MDHAR) (**b**) in the cytosolic fraction of artichoke control plants (Ctrl), mycorrhizal plants (M), plants inoculated with *Verticillium dahliae* (I), and in mycorrhizal plants inoculated with *V. dahliae* (MI). For GR activity, 1 U = 1 nmol of NADPH oxidized min<sup>−</sup>1; for MDHAR activity, 1 U = 1 nmol of NADH oxidized min<sup>−</sup>1; prot. = proteins. The results are given as the mean values of at least five experiments ± SD; \* \* indicates values significantly different from the control (Ctrl) by the Student's *t* test with *p* < 0.01; and - indicate values significantly different from the artichoke inoculated with *V. dahliae* (I) by the Student's *t* test with *p* < 0.05 and 0.01, respectively.

Catalase activity was also analyzed aiming to investigate the ability of mycorrhizal plants to detoxify hydrogen peroxide. No significant changes in CAT activity were found between M and Ctrl plants, as shown in Figure 5 (Panel a). In contrast, a high significant (*p* < 0.01) decrease was observed in MI compared to I. This evidence was confirmed by native electrophoresis where I treatments showed a higher band intensity compared to the MI treatment (Figure 5, Panel b).

**Figure 5.** Spectrophotometric activity (**a**) and electrophoretic profile (**b**) in the cytosolic fraction of catalase (CAT) in artichoke control plants (Ctrl), in mycorrhizal plants (M), in plants inoculated with *Verticillium dahliae* (I), and in mycorrhizal plants inoculated with *V. dahliae* (MI);1U=1 nmol H2O2 dismutated min−1; prot. = proteins. The results are given as the mean values of at least five experiments ± SD; - indicates values significantly different from the artichoke inoculated with *V. dahliae* (I) by the Student's *t* test with *p* < 0.01.

Regarding SOD analysis, the spectrophotometric assay (Figure 6, Panel a), the electrophoretic pattern (Figure 6, Panel b), and the densitometric analysis of the band intensity of Native-PAGE (Figure 6, Panel c), showed a significant increase in its activity in both mycorrhizal plants (M and MI), as compared to control and I plants, respectively. This increase is not due to new additional bands, as three isoforms in all samples have been observed.

**Figure 6.** Spectrophotometric analysis (**a**), electrophoretic pattern of Native-PAGE (**b**), and related densitometric analysis (**c**) in the cytosolic fraction of superoxide dismutase (SOD) of artichoke control plants (Ctrl), mycorrhizal plants (M), plants inoculated with *Verticillium dahliae* (I), and in mycorrhizal plants inoculated with *V. dahliae* (MI); 1 U= the amount of enzyme required to inhibit the reduction rate of NBT by 50% at 25 ◦C; prot. = proteins; a.u. = arbitrary units. The results are given as the mean values of at least five experiments ± SD; \* \* and - indicate values significantly different from the control (Ctrl) and from the artichoke inoculated with *V. dahliae*, respectively, by the Student's *t* test with *p* < 0.01.

### *3.4. H2O2 Content and Lipid Peroxidation Assay*

The effect of mycorrhizal inoculation on ROS accumulation and preservation of membrane structure from oxidative damages after biotic interaction was evaluated by the estimation of H2O2 content and level of lipid peroxidation. The H2O2 content was significantly decreased (*p* < 0.01) in MI treatment with respect to the plants inoculated only with the pathogen (Figure 7, Panel a). Similarly, the mycorrhizal plants inoculated or not with the pathogen showed a decreased level of lipid peroxidation as compared to the inoculated and the control plants, respectively (Figure 7, Panel b).

**Figure 7.** (**a**) Intracellular H2O2 content in artichoke control plants (Ctrl), in mycorrhizal plants (M), in plants inoculated with *Verticillium dahliae* (I), and in mycorrhizal plants inoculated with *V. dahliae* (MI). The results are given as the mean values of at least five experiments ± SD; \* indicates values significantly different from the control (Ctrl) by the Student's *t* test with *p* < 0.05; - indicates values significantly different from the artichoke inoculated with *V. dahliae* (I) by the Student's *t* test with *p* < 0.01; (**b**) Lipid peroxidation level in artichoke control plants (Ctrl), in mycorrhizal plants (M), in plants inoculated with *V. dahliae* (I), and in mycorrhizal plants inoculated with *V. dahliae* (MI). The results are given as the mean values of at least five experiments ± SD; \* and indicate values significantly different from the control (Ctrl) and from the artichoke inoculated with *V. dahliae* (I), respectively, by the Student's *t* test with *p* < 0.05.

### **4. Discussion**

Ensuring stable crop yields and quality while simultaneously guarding human health and the environment is a current challenge facing the farming and research communities. In recent years, the inoculation of plants with AMF has received increasing attention as an environment-friendly approach for improving plant nutrition by increasing nutrients and water availability, nutraceutical values by inducing changes in secondary metabolism, and plant tolerance to biotic and abiotic stress by selecting resistant cultivars and enhancing the activity of antioxidant enzymes [51–56]. According to this view, the present study evaluated the effectiveness of the AMF *G. viscosum* as a biocontrol agent against the soilborne pathogen *V. dahliae* by investigating the antioxidant responses and the effects on ROS metabolism in artichokes. There is sufficient evidence in the literature to confirm that the effect of AMF varies with respect to the host plant and the fungal species [27,57–60]. Based on previous results where *G. viscosum* showed a better affinity with artichoke plantlets in terms of plant growth and physiological activities, compared with *G. intraradices* [61], we selected the former AMF in this study. Our results showed that inoculation with *G. viscosum* significantly improved productivity and ameliorated the disease severity in most of the AMF-treated plants. This is consistent with previous observations showing a beneficial effect on productivity and disease severity in cotton plants affected by Verticillium wilt inoculated with *Rhizophagus irregularis* [62], in wheat plants infected with *Fusarium pseudograminearum* and colonized by *Rhizophagus intraradices* [56], and in potato contaminated by the pathogen *Fusarium sambucinum* and inoculated with the AMF *Glomus irregular* [63]. Although *G. viscosum* had a significant effect on reducing symptoms' development on leaves, the inoculation still caused slight stem browning in most of the AMF-treated plants, confirming previous findings where several AMF treatments showed differences in efficiency towards reducing disease severity [57,64,65].

The findings of the present study highlighted that the mycorrhization process modulated the activity of the ascorbate–glutathione cycle enzymes, including APX, MDHAR, and GR, and the resulting levels of ASC and GSH. In particular, increased levels of GSH and ASC, along with enhanced activity of APX, GR, and MDHAR, were observed in mycorrhizal artichoke plants compared with non-mycorrhizal controls, while the activity of DHAR, as well as the DHA and GSSG contents, remained unchanged. The effects of AMF

on the modulation of ASC-GSH cycle enzymes and antioxidative metabolism have been scarcely studied [66]. Moreover, most of the studies focused on a narrow range of fungi, such as *Trichoderma harzianum* [67], *R. intraradices* [56,68], *Glomus* spp. [69], and the AMF Rhizolive consortium [37]. Previous studies have established a correlation between the level of GSH and resistance to different biotic challenges, including plant viruses, bacteria, and fungi [66,70,71]. Furthermore, GSH represents a key metabolite in the cellular redox buffering system, protecting proteins from irreversible modifications that can be induced by oxidation, through the S-glutathionylation, a post-transcriptional protein modification, which consists in the formation of a stable mixed disulfide between GSH and a protein thiol [72,73]. In this study, the increase in GSH content, observed in both mycorrhizal treatments (M and MI), could be correlated to the higher GR enzyme activity that regenerates the GSH from GSSG, using NADPH as electron donor [74]. Due to the GSH increase, the GSH/GSSG ratio shifts toward the reduced form. Furthermore, the higher availability of NADP+ allows for accepting electrons from photosynthetic electron transport, mitigating the reduction of molecular oxygen to superoxide anion [75]. Moreover, we hypothesize that the increased levels of GSH, ASC, and GSH-dependent enzymes were related with increased mineral elements content (N, P, K, Mg, Fe, Ca, etc.), as demonstrated in previous studies where the higher activity of several antioxidant enzymes was often associated with mycorrhiza-induced increases in biomass and P or N contents [76]. On the other hand, a rise in GSH content is correlated to a higher rate of assimilation of nitrogen and sulfur [77], which are elements of the chemical structure of glutathione.

The increased content of ASC observed in MI plants compared to I, along with the significant increase in MDHAR, corroborates the key role played by MDHAR in the regeneration of ASC from MDHA for ROS scavenging. Moreover, the regeneration of ASC from its oxidative state prevents the intracellular accumulation of DHA that, at high concentrations, has been proved to be toxic for cell metabolisms [78] and to inhibit the activity of enzymes regulated by the thioredoxin–thioredoxin system [79,80]. Our results suggest that MDHAR showed a higher specific activity than DHAR, as reported in previous studies [81–83].

The remarkable increase in H2O2 and MDA in I plants compared with the other treatments was an indicator of oxidative stress caused by pathogen attack. Conversely, in the MI plants, we observed a strong decrease in the H2O2 level that could be explained by the significant increase in the APX enzyme activity of mycorrhizal plants (MI and I) compared to the other treatments. Similarly, decreased MDA concentrations in leaves of AMF, although at a lesser extent, have been observed. These findings are consistent with published results reporting the antioxidant responses in *Digitaria eriantha* plants inoculated with the AMF *R. irregularis* and subjected to drought, cold, or salinity [51] and the effect of AMF on leaf water potential, solute accumulation, and oxidative stress in soybean plants subjected to drought stress [84]. Furthermore, our findings showed a higher level of H2O2 in mycorrhizal plants compared to the non-mycorrhizal plants that is consistent with previous results showing that, in the early stages, the establishment of mycorrhizal symbiosis leads to an increase in ROS content followed by an enhanced defense response of the antioxidant system [26,37,62,85,86]. Accordingly, our results showed a significant stimulation of SOD and APX activity in AMF compared to non-AMF samples, indicating lower oxidative damage in the colonized plants. Conversely, the CAT showed a negative response to AMF, while its activity increased in plants inoculated with pathogen. CAT and APX activities are both involved in the scavenging of H2O2. Although mycorrhizal colonization has been associated with higher antioxidant enzyme activities, the response of the individual enzymes varies with respect to the host plant and the fungal species [58,60]. Moreover, previous studies showed that APX has a much higher affinity for H2O2 than CAT [87–90], while high concentrations of H2O2 induced the expression of genes involved in the synthesis of catalase gene to higher levels, and in less time, than lower H2O2 concentrations [91]. Furthermore, CAT, APX, and SOD are metalloenzymes depending on micronutrients' availability, so their activities may be related to the acquisition of Fe,

Cu, N, P, and Mn in the plants [58,76]. The activity of SOD, APX, and CAT was also analyzed by using electrophoretic systems. Our results showed the presence of three constitutive isoforms in SOD activity in all samples with an enhanced activity in both mycorrhizal plants, and no induction of new isoforms was detected in plants inoculated with the pathogen (I) or in the mycorrhizal plants. Similarly, three SOD isoforms were found in non-mycorrhizal plants of pepper roots affected by Verticillium wilt, although the colonization with the AMF *Glomus deserticola* induced two new isoforms with similar mobility [85].

Overall, our results demonstrated the protective role of the AMF *G. viscosum* on artichoke plants affected by Verticillium wilt through reducing disease severity and enhancing antioxidant systems and activity of investigated antioxidant enzymes. The results of this study suggest that: (1) *G. viscosum* enhances disease tolerance in artichokes; (2) the ascorbate-glutathione cycle plays a key role in maintaining redox balance and avoiding oxidative damage in contaminated artichoke plants inoculated with *G. viscosum*; (3) *G. viscosum* increases the activity of some antioxidant enzymes, such as APX and SOD, while it decreases the activity of some others (CAT), confirming that the efficiency of AMF is related to fungal and/or plant species, soil nutrient availability, and environmental factors. All those data can lend support to the applications of AM *G. viscosum* as a costeffective and environment-friendly strategy for reducing or alleviating *V. dahliae* effects in artichoke plants.

**Author Contributions:** Conceptualization, C.P. and A.V.; methodology, C.P. and A.V.; data curation, C.P. and A.V.; writing—original draft preparation, C.P. and A.V.; writing—review and editing, C.P., A.V. and F.T.; supervision, C.P.; funding acquisition, C.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by University of Bari Aldo Moro, grant number H96J15001610005.

**Acknowledgments:** The authors thank Franco Ciccarese for providing plant materials used in this study.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**


## *Article* **Fungal Associates of Soft Scale Insects (Coccomorpha: Coccidae)**

**Teresa Szklarzewicz 1, Katarzyna Michalik 1, Beata Grzywacz <sup>2</sup> , Małgorzata Kalandyk-Kołodziejczyk <sup>3</sup> and Anna Michalik 1,\***


**Abstract:** *Ophiocordyceps* fungi are commonly known as virulent, specialized entomopathogens; however, recent studies indicate that fungi belonging to the Ophiocordycypitaceae family may also reside in symbiotic interaction with their host insect. In this paper, we demonstrate that *Ophiocordyceps* fungi may be obligatory symbionts of sap-sucking hemipterans. We investigated the symbiotic systems of eight Polish species of scale insects of Coccidae family: *Parthenolecanium corni*, *Parthenolecanium fletcheri*, *Parthenolecanium pomeranicum*, *Psilococcus ruber*, *Sphaerolecanium prunasti*, *Eriopeltis festucae*, *Lecanopsis formicarum* and *Eulecanium tiliae*. Our histological, ultrastructural and molecular analyses showed that all these species host fungal symbionts in the fat body cells. Analyses of ITS2 and Beta-tubulin gene sequences, as well as fluorescence in situ hybridization, confirmed that they should all be classified to the genus *Ophiocordyceps*. The essential role of the fungal symbionts observed in the biology of the soft scale insects examined was confirmed by their transovarial transmission between generations. In this paper, the consecutive stages of fungal symbiont transmission were analyzed under TEM for the first time.

**Keywords:** soft scale insects; *Ophiocordyceps*; symbiosis; transovarial transmission

### **1. Introduction**

Scale insects (coccoids) are plant sap-sucking hemipterans that are considered serious pests in agriculture, horticulture, and forestry. These insects cause direct damage to plants through sap-sucking and the injection of toxic saliva into plant tissue, which is a cause of the retardation of plant growth and recovery, and furthermore, may lead to the death of the whole or part of the plant if the infestation is severe. Scale insects are rarely known as vectors of bacterial pathogens or phytoplasmas, and only a few species are involved in virus transmission [1]. Most species of scale insects also cause indirect damage by producing a carbohydrate-rich solution, referred to as honeydew, which is a medium for the growth of saprophytic fungi known as "sooty molds", forming black superficial colonies that also reduce the host plant photosynthesis rates, further diminishing the vigor of the plant (e.g., [1–3]).

Scale insects are highly diverse in terms of the morphology of their external and internal organs, reproductive strategies and chromosome systems, as well as symbiotic systems, which makes them an interesting group of insects to study [4]. After the Diaspididae and Pseudococcidae, the Coccidae (soft scales, coccids) is the third largest family of scale insects in terms of species richness. There are 1281 described species of coccids in 176 genera. Soft scales are widely distributed in all zoogeographical regions; however, they predominantly occur in the tropics and subtropics [2,5–7]. The Coccidae, like other scale insect families, exhibit a remarkable dimorphism. The adult females are wingless

**Citation:** Szklarzewicz, T.; Michalik, K.; Grzywacz, B.; Kalandyk -Kołodziejczyk, M.; Michalik, A. Fungal Associates of Soft Scale Insects (Coccomorpha: Coccidae). *Cells* **2021**, *10*, 1922. https://doi.org/10.3390/ cells10081922

Academic Editor: Suleyman Allakhverdiev

Received: 28 May 2021 Accepted: 26 July 2021 Published: 29 July 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

and lack a well-defined head, thorax, and abdomen. The adult males are usually winged, with distinct body parts, and do not possess functional mouthparts. A large number of soft scale species are notorious plant pests that are of great economic importance to crops. Many pests of the Coccidae have been introduced into new zoogeographical regions, thus making them cosmopolitan [2].

In several insect groups, including scale insects, a mutualistic relationship with microorganisms (bacteria or fungi) evolved. The use of genomic analyses has confirmed earlier assumptions that the occurrence of symbiotic associates in the insect body is associated with the poor diet of the host-insect, e.g., plant sap-sucking hemipterans receiving amino acids, and blood-sucking insects receiving B vitamins from their mutualists [8].

As typical phloem-feeders, scale insects live in symbiotic association with microorganisms; however, in comparison with close relatives such as aphids, whiteflies, and psyllids, these insects are characterized by highly diverse symbiotic systems. Scale insects may live in mutualistic relationships with different species of bacteria or fungal symbionts. They may have only one symbiont or several species of microorganisms. Symbionts may be harbored in the fat body cells, in the midgut epithelium, in the specialized cells of mesodermal origin termed bacteriocytes, or inside the cells of other bacteria. Scale insects also developed different modes of transmission of their symbiotic associates from mother to offspring [9–12].

In contrast to other families of scale insects, symbionts of soft scale insects have not been as extensively examined through the use of modern ultrastructural and molecular methods. The results of histological studies (reviewed in [9,10,13]) have indicated that these insects are hosts to obligate fungal symbionts that may be localized freely in hemolymph or intracellularly in fat body cells, and are transovarially inherited. Studies recently conducted with the use of molecular methods and phylogenetic analyses have allowed the identification of the symbiotic associates of seven species of the Coccidae from the Mediterranean region and 28 species from southern China as the *Ophiocordyceps*-allied fungus (phylum Ascomycota) [14,15]. It is noteworthy that, for many years, fungi belonging to the genus *Ophiocordyceps*, like other members of Ophiocordycypitaceae and Cordycypitaceae, were known mainly as entomopathogenic microorganisms [16,17]. They may attack various species of insects, e.g., ants, beetles, butterflies, and hemipterans. The hyphae of these fungi penetrate the body wall and destroy their internal tissue: fat body cells, hemocytes, muscle, nerve ganglions, and the intestine. In each case, insects infected by these fungi die before beginning their reproductive phase, i.e., within 48–96 h of penetration [16]. For this reason, entomopathogenic fungi have also been tested as biological control agents for whiteflies, lepidopterans and scale insects [18,19]. The finding of a close relationship between fungal entomopathogens and symbionts has led to the hypothesis that during their co-evolution, the interaction between both of these partners shifted from parasitism to mutualism [20,21].

The aim of this study was to further explore the symbiotic systems of the Coccidae family: (1) to determine the systematic position of symbionts, (2) to verify whether symbiosis with fungi is a general rule of this family, (3) to show whether the symbiosis is the result of the single infection of the ancestor of Coccidae or multiple independent infections, (4) to describe symbiont distribution and ultrastructure as well as a mode of transmission from mother to progeny in eight species from three subfamilies of Central European origin.

### **2. Material and Methods**

### *2.1. Insects*

Eight species of the Coccidae family: *Parthenolecanium corni* (Bouché, 1844), *Parthenolecanium fletcheri* (Cockerell, 1893), *Parthenolecanium pomeranicum* (Kawecki, 1954), *Eriopeltis festucae* (Boyer de Fonscolombe, 1834), *Lecanopsis formicarum* Newstead, 1893, *Sphaerolecanium prunastri* (Boyer de Fonscolombe, 1834), *Eulecanium tiliae* (Linnaeus, 1758) and *Psilococcus ruber* Borchsenius, 1952 were collected in unprotected areas in Poland between the years 2017 and 2019 from their host plants. The localities, collection dates, and host

plants of the investigated species have been summarized in Table 1. Species of the Coccidae family were assigned to subfamilies according to Koteja [22].



### *2.2. Light (LM) and Electron Microscopy (TEM)*

Females of the investigated species, destined for detailed histological and ultrastructural analysis, were fixed in 2.5% glutaraldehyde in 0.05 M phosphate buffer (pH 7.4) and stored in a fridge for 1–4 weeks. After this time, the samples were rinsed five times in the buffer with sucrose (5.8 g/100 mL), postfixed in buffered 1% osmium tetroxide for 2 h, and then dehydrated in an ethanol series (30%, 50%, 70%, 90%, 100%) and acetone. Finally, the samples were embedded in epoxy resin Epon 812 (Serva, Heidelberg, Germany) ([23] modified). For the histological analyses, semithin sections (1 μm thick) were stained in 1% methylene blue in 1% borax and photographed under a Nikon Eclipse 80i light microscope. For ultrastructural analyses, ultrathin sections (90 nm thick) were doubly contrasted with uranyl acetate and lead citrate and subsequently examined and photographed under a Jeol JEM 2100 at 80 kV transmission electron microscope. The number of specimens used for histological and ultrastructural analyses is summarized in Table 1.

### *2.3. Molecular Analyses*

Specimens destined for molecular analysis were fixed in 100% ethanol. In the case of the removal of the surphase's contaminations, the specimens were placed in 5% sodium hypochlorite for 1 min and then rinsed in distilled water three times for one minute. Then the cuticle was removed, and the DNA isolated only from the fat body and internal organs. DNA extraction was performed separately from 3-7 individuals of each species (see Table 1) using the Bio-Trace DNA Purification Kit (EURx, Gda ´nsk, Poland) following manufacturer protocol and subsequently stored at −20 ◦C for further analysis.

The fungal associates of the species examined were identified and characterized based on sequences of two genes: Internal Transcribed Spacer 2 of nuclear ribosomal RNA (ITS 2) and Beta-tubulin gene using primers: ITS3/ITS4 [24] and Ophi\_Btub44448F/Ophi\_Btub5243R (D. Vanderpool, unpublished), respectively. The mitochondrial cytochrome c oxidase subunit I (COI) gene of soft scale insects was amplified using the primer pair PCoF1 and HCO [25]. The conditions for all the PCR reactions were an initial denaturation step at 94 ◦C for 5 min, followed by 33 cycles at 94 ◦C for 30 s, Tm for 40 s, 70 ◦C for 1 min 40 s and a final extension step of 5 min at 72 ◦C. The PCR products were visualized on 1.5% agarose gel stained with Simply Safe (EURx, Gda ´nsk, Poland), purified with the Gel-Out Concentrator (A&A Biotechnology, Gda ´nsk, Poland) kit following manufacturer protocol, and subsequently sequenced. The sequences of the primers have been listed in Table S1. The nucleotide sequences obtained were deposited in the GenBank database under the accession numbers: ITS (MN733271-MN733277, MZ594469); COI (MN603157, MN603159-MN603160, MN603162-MN603164, MZ567176), Beta-tubulin (MN750822-MN750828, MZ576194).

### *2.4. Fluorescence In Situ Hybridization (FISH)*

Fluorescence in situ hybridization (FISH) was conducted with the probe Hyp760 specific for the 18S rRNA gene of *Ophiocordyceps* fungi [26] (Table S1). Two individuals of each species that were preserved in 100% ethanol were rehydrated, fixed in 4% formaldehyde for two hours and dehydrated through incubation in 80%, 90% and 100% ethanol and acetone. The material was then embedded in Technovit 8100 (Kulzer, Werheim, Germany) resin and subsequently cut into sections. Hybridization was performed using a hybridization buffer containing: 1 mL1MTris-HCl (pH 8.0), 9 mL 5 M NaCl, 25 μL 20% SDS, 15 mL 30% formamide and about 15 mL of distilled water. The slides were incubated in 200 μL of hybridization solution (hybridization buffer + probes) overnight at room temperature [27]. Following this, the slides were washed in PBS three times for 10 min, then dried and covered with a ProLong Gold Antifade Reagent (Life Technologies, Carlsbad, CA, USA). The hybridized slides were then examined using a confocal laser scanning microscope Zeiss Axio Observer LSM 710.

### *2.5. Phylogenetic and Co-Phylogenetic Analyses*

The sequences were aligned in a CodonCode Aligner v.8.0 (CodonCode Corporation, www.codoncode.com, 8 March 2018). Coding regions were translated to amino acids using Mega v. X [28] in order to detect frameshift mutations and internal stop codons. The Akaike Information Criteria (AIC) in MrModeltest v. 2.2 [29] was used to estimate the best-fit substitution models. Phylogenetic trees were constructed using Bayesian inference (BI) in MrBayes v. 3.2.6 [30]. For phylogenetic trees of both scale insects and fungal symbionts, MrBayes was run for six million generations, sampling every 100 generations in order to ensure the independence of the samples. Two independent runs were performed to ensure that convergence on the same posterior distribution was reached, and if the final trees converged on the same topology. The statistical confidence in the nodes was evaluated using posterior probabilities.

Co-phylogenetic and host-switching events were tested in Jane v.4 [31] using the BI host and fungal trees as input. The analysis was performed with 100 generations, population sizes of 100 and a default event–cost scheme including "co-speciation", "duplication", "host switch", "losses", and "failure to diverge".

### **3. Results**

### *3.1. Fungi Belonging to the Ophiocordycypitaceae Family Are Symbionts of the Soft Scale Insects Examined*

Our histological, ultrastructural, and molecular analyses showed that all the species examined were associated with symbiotic fungi. Molecular analyses based on sequences of ITS2 and Beta-tubulin genes revealed that in all the species examined, the symbiotic fungi belonged to the Ophiocordycypitaceae family within the Ascomycota phylum (Ascomycota: Sordariomycetes: Hypocreales: Ophiocordycipitaceae).

Based on the sequences of the Beta-tubulin gene, two groups of symbiotic microorganisms may be distinguished: the first one includes symbionts of *Eulecanium tiliae*, *Parthenolecanium corni*, *Parthenolecanium pomeranicum*, and *Parthenolecanium fletcheri*, and the second one is comprised of the symbionts of remaining species. These latter sequences are almost

identical (99%) and differ from the sequences in the first group by 4%. In turn, the similarity of the ITS2 sequences, which are more species-specific, ranges from 87% to 96%. Blast searches for all of the sequences obtained demonstrated the highest similarity to sequences of homologue genes of various species of *Ophiocordyceps* or its anamorphic (i.e., asexual) form—*Hirsutella*. Phylogenetic analyses also confirmed the systematic affiliation of *Hirsutella* with the genus *Ophiocordyceps*, and showed that they create a sister group, i.e., *O. cochlidiicola*, that is closely related to *H. leizhouensis*, while *O. arborescens*, *H. versilor*, and *O. xuefengensis* are closely related to *H. illustris* (Figure 1 and Figure S1). The co-phylogenetic analysis based on the ITS2 gene of fungal symbionts and the COI genes of host scale insects returned 11 potential co-speciation events, one duplication, 22 duplications with host switch, five losses, and one failure to diverge (Figure 2).

**Figure 1.** Phylogenetic tree showing the relationships between fungal symbionts of soft scale insect species, pathogenic, as well as free living fungi (constructed on the base of ITS2 genes). The numbers near the nodes refer to the Bayesian posterior probability.

**Figure 2.** Co-phylogenetic analysis between the *Ophiocordyceps* symbionts' tree and their host's tree (constructed on the base of the ITS2 genes of fungal symbionts and COI genes of hostscale insects). Black and blue lines indicate the phylogenies of the scale insects and *Ophiocordyceps,* respectively. Hollow red circles indicate co-speciation events, solid red and yellowcircles indicate duplications, arrows indicate host events, dashed lines indicate losses, and uneven lines indicate failures to diverge.

The analysis of serial semithin sections has shown that symbiotic fungi are distributed only within the fat body cells (Figure 3). They were not observed in any other tissue except the ovaries, which is related to the transovarial transmission of these symbionts between generations (see the Results subsection, which follows). The number of symbionts and their density in the host insect body are subfamily specific and are not dependent on the stage of the insect's development (Figure 3). We observed the same amount of cells of fungi in the body cavities of larvae and mature females. The smallest number of symbionts was observed in the representatives of the Eriopeltinae subfamily (with the exception of *Psilococcus ruber*), where only single groups of fungi occur (Figure 3A–C). The highest density was observed in the members of Filippinae subfamily (Figure 3G–J). In all representatives of the Filippinae subfamily examined: *Sphaerolecanium prunasti* and *Eulecanium tiliae*, all cells of the fat body are filled with numerous symbiotic fungi (Figure 3G–J). The ultrastructural analyses showed that the cells of fungi are surrounded by a thick cell wall (Figure 3B,E,H). Large nuclei (Figure 3E,H) and vacuoles (Figure 3B) are visible in their cytoplasm.

**Figure 3.** Distribution of symbiotic fungi in the body cavity of species examined (**A**–**J**). *Ophiocordyceps* fungi in the cytoplasm of the fat body cells (**A**,**D**,**G**). Light microscope, scale bar = 20 μm (**B**,**E**,**H**). TEM, scale bar = 2 μm (**C**,**F**,**I**,**J**). Confocal microscope, scale bar = 20 μm (**A**–**C**). Eriopelitnae subfamily (**A**). *Eriopeltis festucae* (**B**,**C**). *Lecanopsis formicarum* (**D**–**F**). Coccinae subfamily (**D**,**E**). *Parthenolecanium corni* (**F**). *Parthenolecanium pomeranicum* (**G**–**J**). Filippinae subfamily, *Sphaerolecanium prunastri*. N—nucleus of the fungal cell; white arrow—fungal symbiont, white arrowhead—nucleus of the fat body cell, black arrow—vacuole.

The presence of *Ophiocodyceps* fungi in the body cavity of the soft scale insects examined was also confirmed by fluorescence in situ hybridization using an *Ophiocordyceps*specific probe (Figure 3C,F,I,J). The microscopic observations did not show any damage to the insects' tissue caused by fungi.

### *3.2. Fungal Associates of Soft Scale Insects Are Transovarially Transmitted between Generations*

Microscopic observations revealed that fungal symbionts residing in the examined species of soft scale insects were inherited transovarially, i.e., they infect female germ cells.

The ovaries of soft scale insects are composed of numerous short telotrophic ovarioles, which are subdivided into an anterior tropharium (trophic chamber) and posterior vitellarium (Figure 4A) (for further details concerning the organization of the ovaries of scale insects, see [32]). The vitellarium houses a single oocyte, which is connected with the tropharium by means of a broad nutritive cord (Figure 4A). The oocyte is surrounded by a single-layered follicular epithelium (Figure 4A). At the time the ovarioles contain the oocytes in the stage of advanced choriogenesis (Figure 4A), the fungal symbionts begin to enter follicular cells surrounding the neck region of the ovariole (i.e., the region between the tropharium and the developing oocyte) (Figure 4B). After crossing through the cytoplasm of the follicular cells (Figure 4A,C,D), symbionts temporarily gather around the nutritive cord (Figure 4E,F). Next, symbiotic microorganisms migrate along the nutritive cord to the space between follicular epithelium and oocyte surface (Figure 4G,H). Finally, symbionts enter the oocyte cytoplasm (Figure 4I,J), where they remain until the beginning of embryonic development.

**Figure 4.** Consecutive stages of symbiont transmission between generations (**A**,**B**). Longitudinal section through the ovariole of *Parthenolecanium fletcheri*. Symbiotic fungi (white arrows) invade the follicular cells in the neck region of the ovariole (**A**). Light microscope, scale bar = 20 μm (**B**). TEM, scale bar = 2 μm (**C**,**D**). Symbiotic fungi in the cytoplasm of follicular cells of *Parthenolecanium pomeranicum* (**C**) and *Eulecanium tiliae* (**D**). (**C**) TEM, scale bar = 2 μm (**D**). Confocal microscope, scale bar = 20 μm (**E**,**F**). Symbiotic fungi surrounding the nutritive cord (**E**). *Parthenolecanium pomeranicum* (**F**). *Parthenolecanium fletcheri* (**E**,**F**). Light microscope, scale bar—20 μm (**G**). Symbiotic fungi move along the nutritive cord to the perivitelline space of *Parthenolecanium fletcheri*. Light microscope, scale bar = 20 μm (**H**). Symbiotic fungi gather in the invagination of the perivitelline space of *Eulecanium tiliae*. Confocal microscope, scale bar = 20 μm (**I**). Migration of symbionts from the perivitelline space to the oocyte cytoplasm of *Psilococcus ruber*. Light microscope, scale bar = 20 μm (**J**). Symbiotic microorganism in the oocyte cytoplasm of *Psilococcus ruber.* TEM, scale bar = 2 μm. f—follicular cell, nc—nutritive cord; oc—oocyte, t—trophocyte, tc—trophic core, tn—trophocyte nucleus, white arrow—fungal symbiont; asterisk—perivitelline space.

### **4. Discussion**

The Cordycypitaceae and Ophiocordycypitaceae families of Ascomycota include several genera that are commonly occurring entomopathogens, such as *Ophiocordyceps*, *Cordyceps*, *Hirsutella*, *Lecanicilium*, and *Metarhizium* [33]. Among them, the *Ophiocordyceps* species, which usually attack ants, are best known for their ability to manipulate ant behavior. However, recent research that applies the use of molecular techniques, and concerns the interactions of insects and various microorganisms indicates that the fungi of Ascomycota phylum may also live in symbiotic associations with insects [14,26,34]. So far, fungal associates have been found and described in many representatives of Hemiptera, Coleoptera, Diptera, and Hymenoptera, as well as in some members of Isoptera, Neuroptera, and Lepidoptera; for a review, see [35]. They belong to various families of Ascomycota; however, hemipterans are usually associated with fungi from the Ophiocordycypitaceae family.

Within the Hemiptera: Auchenorrhyncha (i.e., Fulgoromorpha (planthoppers) and Cicadomorpha (leafhoppers, treehoppers, spittlebugs, and cicadas)), the presence of *Ophiocordyceps*-allied symbionts was confirmed in some leafhoppers from the Deltocephalinae subfamily [36,37] and planthoppers from the Flatidae and Delphacidae families [38–40], as well as in Japanese cicadas [26]. In Hemiptera: Sternorrhyncha, the symbiosis with fungi is not as common as in Auchenorrhyncha, and has so far only been observed in some of the Hormaphidinae aphids and members of the Coccidae, Dactylopiidae, and Kermesidae families of scale insects [14,15,34,36,41–43].

In this paper, using microscopic and molecular techniques, we investigated symbiotic systems of soft scale insects (Coccidae) belonging to three subfamilies: Coccinae, Eriopeltinae and Filippinae. Our analyses revealed that all of the species investigated were only host to fungal symbionts. Analyses of sequences of ITS2 as well as Beta-tubulin genes demonstrated that these microorganisms are representatives of the Ophiocordycypitaceae family. However, the ITS sequences, which are more species-specific than the Beta-tubulin gene, display about 87–95% similarity to each other. Fungal symbionts of members of the Coccidae family have previously been studied by Gomez-Polo and co-workers [14] and Deng and co-workers [15]. These authors investigated seven coccid species from the Ceroplastinae and Coccinae subfamilies collected in Spain, Israel, and Cyprus. Based on a high-throughput sequencing of ribosomal genes, these authors showed that the species analyzed were mainly associated with *Ophiocordyceps* fungi.

It is believed that the occurrence of fungal associates in some auchenorrhynchans and some aphids is a result of symbiont replacement [26,36,37], e.g., in deltocephalinae leafhoppers as well as in Japanese cicadas, fungi replaced the bacteria *Nasuia* and *Hodgkinia* (respectively), in Delphacidae and Flatidae planthoppers – bacteria *Sulcia* and *Vidania,* in the aphids of Hormaphidinae subfamily–bacteria *Buchnera* [26,36,37,41,44–47]. Our phylogenetic analyses showed that *Ophiocordyceps*-allied fungi in the species of soft scale insects examined form a clade (see Figure 1), which suggests that symbiosis between these insects and their microorganisms is the result of a single infection of the ancestor of extant coccids. However, the differences observed in ITS2 sequences (5–13%) may indicate their independent evolution after the initial infection.

Since symbiotic fungi have recently been observed in various hemipteran lineages, researchers continually ask themselves about the origin of these associations [14,20,34,35,40]. The literature data indicate three possible evolutionary scenarios: (1) symbiotic fungi may derive from entomopathogenic fungi; (2) they may be the descendants of nonpathogenic commensals; or (3) the ancestor of fungal symbionts may be phytopathogenic fungi [21,35,38]. Most hemipterans are plant sap-sucking insects, and due to their mode of feeding, they are also vectors of plant pathogens. Therefore, it seems probable that they may acquire fungal symbionts from the host plant. However, the results of molecular phylogenetic analyses that indicate the close relationship between fungal symbionts of hemipterans with entomopathogenic fungi favor the concept that the ancestors of the present fungal symbionts were entomopathogens that lost their virulence and shifted to a symbiotic lifestyle. The genomic analysis of the fungal symbiont of the planthopper

*Nilaparvata lugens* showed that it possesses a smaller genome than its free-living relatives and does not possess genes encoding enzymes responsible for penetrating the insect's cuticle, solubilizing its tissue, or genes related to sexual reproduction [40].

*Ophiocordyceps* fungi, in the soft scale insects examined, are localized in the cytoplasm of the fat body cells. The same localization of the fungal symbiont was observed in other Coccidae species that have previously been examined, in the scale insect *Kermes quercus* (Kermesidae) and leafhoppers *Fieberiella septentrionalis*, *Graphocraerus ventralis* and *Orientus ishidae* [9,14,34,37]. In the Deltocephalinae leafhopper *Cicadula quadrinotata*, fungal symbionts were found to be present in the cytoplasm of midgut epithelium cells, in fat body cells, and free in the hemolymph [37]. In contrast, in Japanese cicadas and planthoppers from the Flatidae and Delphacidae families, they are harbored in the cells of a specialized host's organs, termed mycetomes [26,47]. It seems probable that, similarly to the case of the bacterial associates of insects [48], the occurrence of fungal symbionts in the digestive tract represents the initial state of colonization of the insect body through microorganisms, in the hemolymph and in fat body cells represents the next (i.e., intermediate) stage, whereas their presence in the cells of mycetomes represents the most advanced condition of this association. It is worth mentioning that the occurrence in the fat body and in the specialized host's cells seems to be characteristic to fungi from the Ophiocordycypitaceae family, whereas other species of fungi found so far in insects are usually localized in a different part of the digestive tract [35].

Scale insects, like other insects living in a mutualistic relationship with microorganisms, develop stable mechanisms of transmission of these associates from one generation to the next. The results of numerous studies conducted both earlier and more recently indicate that scale insects are not only characterized by diverse species and distributions of symbionts, but also by different modes of transmission to their progeny ([9,12,49–58], this study). It should be stressed that even members of the same family may inherit symbionts differently (for further details, see [58]). It should be stressed that scale insects (i.e., all the Pseudococcidae, Eriococcidae, Coccidae, and Putoidae examined so far) are unique in that they are the only group of insects in which microorganisms invade the neck region of the ovariole ([50,55–57], this study). Until now, the course of transmission of fungal symbionts in soft scale insects had not been studied under TEM; however, Gomez-Polo and co-workers [14] reported their presence in eggs, and thus proved that these microorganisms are transported between generations transovarially. Our observations of the ovaries of the members of the Coccidae family showed that their fungal associates are transmitted between generations similarly to bacterial symbionts in Pseudococcidae, Eriococcidae, and Putoidae [50,53,55–57]. One noteworthy aspect is that, just as in the case of the infestation of ovarioles through bacterial symbionts, the time of transmission of the fungal symbionts in soft scale insects is correlated with the stage of oogenesis—the microorganisms commence the infection of the ovarioles that contain the oocytes in the stage of advanced choriogenesis. Similarly to Pseudococcidae, Eriococcidae, and Putoidae, in Coccidae, symbionts invade follicular cells surrounding the nutritive cord, because this area is the only place on the oocyte surface that is devoid of egg envelopes (see Figure 4A). After the degeneration of the nutritive cord, the symbionts may enter the oocyte cytoplasm.

Numerous molecular analyses have confirmed that bacterial symbionts co-evolve with their host insects [59–61]. The co-diversification of fungal associates and insects has previously not been tested intensively. Our co-phylogenetic analysis, through the use of Jane software, indicated that not all the species of soft scale insects examined co-evolved with their host (see Figure 2). It is a noteworthy fact that Gomez-Polo and co-workers [14] additionally revealed that co-phylogeny of Coccidae, which they tested, and their *Ophiocordyceps* symbionts were incongruent. These authors suggested that this incongruence may be a result of the independent acquisition of fungi by particular members of Coccidae. However, taking into account the fact that some of the species examined coevolved with their symbiotic partners (this study), it may be speculated that incongruence in the co-phylogeny of some coccids and their fungal associates may result from the

independent evolution of fungal symbionts or their replacement during the evolutionary history of different species.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/cells10081922/s1. Figure S1. Phylogenetic tree showing the relationships between fungal symbionts of soft scale insect species, pathogenic, as well as free-living fungi (constructed on the base of sequences of Beta-tubulin gene). The numbers near the nodes refer to the Bayesian posterior probability. Table S1. List of primers and fluorochrome-labeled probe used in this study.

**Author Contributions:** Conceptualization, A.M. and T.S.; methodology, A.M.; software, B.G.; investigation, A.M., K.M.; resources, M.K.-K.; data curation, A.M.; writing—original draft preparation, A.M., M.K.-K., T.S.; writing—review and editing, A.M., M.K.-K., T.S., B.G., K.M.; visualization, A.M., B.G., T.S.; supervision, A.M., T.S.; project administration, A.M.; funding acquisition, A.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the Iuventus Plus V grant IP2015050374 from the Ministry of Science and Higher Education to AM and N18/DBS/000013 to Jagiellonian University.

**Institutional Review Board Statement:** According to Polish Act on the Protection of Animals used for Scientific or Educational Purposes, studies on unprotected insects do not require any permission.

**Informed Consent Statement:** This article do not contain research involving humans.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** We are greatly indebted to Ada Jankowska for her skilled technical assistance. Ultrastructural observations were carried out using the Jeol 2100 transmission electron microscope in the Laboratory of Microscopy, Department of Cell Biology and Imaging, Institute of Zoology and Biomedical Research, Jagiellonian University. The open-access publication of this article was funded by the Priority Research Area BioSunder the program "Excellence Initiative–Research University" at the Jagiellonian University in Kraków.

**Conflicts of Interest:** T.S., K.M., B.G., M.K.-K. and A.M. declare no conflict of interest.

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