Antifungal activity of local isolates of Beauveria bassiana (Balsamo) Vuillemin against Verticillium dahliae Kleb. causing wilt disease of cotton

Abstract

Background

Verticillium dahliae Kleb. is a soil-borne pathogen with a broad host spectrum and causes yield losses in cotton cultivation worldwide. Beauveria bassiana is an environmentally friendly entomopathogenic fungus (EPF) that has been recognized, used and studied for many years.

Results

As a control, all local of B. bassiana isolates inhibited mycelial growth of the pathogen at different rates. The highest percentage of inhibition against non-defoliating (PHCVd3 isolate) and defoliating (PHCVd47 isolate) pathotypes was determined in Bb18 (85.3; 81.7%) and Bb1 (82.3; 77.5%) isolates, respectively, when applied 2 days ago. Isolates Bb1 (80.6%) and Bb18 (77.8%) had the highest percentages of inhibition against the PHCVd3 isolate, and Bb18 (75.8%) against isolate PHCVd47, when EPF and pathogen were applied at the same time.

Conclusions

Bb18 and Bb1 isolates of B. bassiana showed a hope against both pathotypes of wilt disease caused by V. dahliae under in vitro conditions. Especially, B. bassiana was observed to induce higher inhibition rates, when EPF isolates developed in Petri dish 2 days before the pathogen.

Background

Cotton (Gossypium spp.) is an industrial crop that, with its widespread and mandatory fields of use, has great economic importance for humanity and creates added value and jobs for producing countries (Majumdar et al. 2019). Turkey ranks sixth in world cotton production after India, China, the USA, Brazil and Pakistan (USDA 2021).

One biotic factor affecting cotton production and quality is wilt disease caused by Verticillium dahliae Kleb. (Fradin and Thomma 2006). V. dahliae is a soil-borne fungal plant pathogen responsible for Verticillium wilt, which affects a variety of plants, including crops, flowers and vegetables (Jimenez-Diaz et al. 2012). This pathogen can persist in the soil through microsclerotia for up to 14 years (Xiao and Subbarao 1998). Globally, the pathogen has two pathotypes, defoliating and non-defoliating. While the defoliating pathotype causes complete shedding and leaf death, the non-defoliating pathotype causes wilting and less leaf destruction (Bejarano-Alcazar et al. 1995). Both pathotypes have been reported to occur in Turkey, with 93% of the defoliating isolates found in the Aegean region and 77% of the non-defoliating isolates found in the Çukurova and Southeast Anatolia (Göre 2007). Cotton yield losses caused by Verticillium wilt average around 10–35% (Song et al. 2020). The pathogen enters by infecting plants through capillary roots and settling in vascular bundles. Then, starting from the lower leaves, it causes wilting, drying, reduced photosynthesis, collapse of small pods, yield loss and changes in fiber quality characteristics (Agrios 2005). Controlling the disease is complicated by the fact that the pathogen is a soil fungus and there is no economical chemical control.

Beauveria bassiana (EPF) (Bals.-Criv.) Vuill. (Hypocreales: Cordycipitaceae) has been observed in more than 700 species (Wraight et al. 2000). B. bassiana is an entomopathogenic fungus (EPF) used for biological control against both plant pathogens and insect pests and the cause of white muscardine (Feng et al. 2004). Today, B. bassiana, Metarhizium anisopliae, Isaria fumosorosea (= Paecilomyces fumosoroseus) and Lecanicillium lecanii are commercially produced among the 700 identified species of EPF (Meyling et al. 2018). EPFs have very important advantages such as being non-toxic to human and environmental health, having a wide host range, not developing host resistance, being used in combination with pesticides, being cheap and easy to use (Sinha et al. 2016). Studies in vivo or in vitro, Beauveria spp. showed antagonistic activity against Botrytis cinerea Pers., Fusarium oxysporum Bad. correct. Snyder & Hansen, Gaeumannomyces graminis (Sacc.) Arx & D.L. Olivier, Pythium spp., Rhizoctonia solani Kühn and Septoria spp. (Dara 2019). Meanwhile, plant pathogen control studies of B. bassiana are, generally, limited to in vitro studies of plant pathogen growth inhibition and cell wall structure degradation. There is a little research on the efficacy of B. bassiana against pathogens under field conditions. B. bassiana has been reported to inhibit the mycelial growth of Rhizoctonia solani and Pythium myriotylum in cotton (Ownley et al. 2008), Botrytis cinerea (Barra-Bucarei et al. 2020).

The aim of the study is to investigate the antifungal activity of 5 local EPF isolates of B. bassiana (ET 10, ET 101, BMAUM-M6-4, Bb1, and Bb18) isolated from different hosts in Turkey against 2 fungal isolates of V. dahliae (PHCVd3-non-defoliating pathotype and PHCVd47-defoliating pathotype) under laboratory conditions.

Methods

Fungal isolates

The EPF isolates of B. bassiana (EPF) and fungal isolates of V. dahliae (pathogen) used in this study are shown in Table 1. Five local isolates of B. bassiana were obtained from different hosts and locations in Turkey. All B. bassiana isolates were developed within the dark at 25 ± 1 °C for 7–15 days and after that subcultured on potato dextrose agar (PDA-Difco). Pure cultures of V. dahliae (PHCVd3 isolate-nondefoliating pathotype; PHCVd47 isolate-defoliating pathotype) were obtained from the collection of fungal cultures at Mustafa Kemal University of Agriculture, Department of Plant Protection, and subcultured in PDA at 25 ± 1 °C, 12 h dark/12 h light with an incubation period of 7–15 days.

Table 1 Sources and origins of the EPF fungus and tested pathogen fungal isolates used in the experiment

Antagonistic potential of B. bassiana against V. dahliae under laboratory conditions

Effect of local EPF isolates of B. bassiana on V. dahliae was investigated in the General Microbiology Laboratory of the Department of Organic Farming Business Management, Faculty of Applied Sciences, Pamukkale University. Antifungal interactions between EPF and test pathogen were determined using an in vitro dual culture technique on PDA medium in 90 mm Petri dishes. In the first experiment, actively growing B. bassiana mycelial disks (5 mm in diameter, 7–15 days old) were placed 3 cm from the corners of Petri dishes poured with PDA (25 mL). B. bassiana was plated, 2 days before V. dahliae due to the high growth rate of it. Two days after inoculation, mycelial disks of the test pathogen (5 mm in diameter, 7–15 days old) were placed next to those of B. bassiana at a distance of 3 cm from the Petri dish. In a second experiment, mycelial disks of B. bassiana and V. dahliae (5 mm in diameter, 7–15 days old) were placed concurrently at a distance of 3 cm from the corner of a Petri dish with PDA. As a control, only a single mycelial disk (5 mm in diameter, 7–15 days old) containing the test pathogen was placed in the center of the Petri dish. Plates were then, sealed with parafilm and incubated at a temperature of 25 ± 1 °C in the dark for the first and second experiments. Experiments were carried out with five replicates in a completely randomized parcels design. When the test pathogen colonized the whole Petri dish, the percentage of inhibition of mycelial growth of the test pathogen was determined according to the method described by Sundaramoorthy et al. (2012) formula (Eq. 1).

$${\text{PI}}: \, \left[ {\left( {{\text{C }} - {\text{ T}}} \right) \, /{\text{ C}}} \right] \, \times { 1}00$$

(1)

where PI is the percentage inhibition of mycelial growth (%), C is the radial growth of the pathogen in control (mm) and T is the radial growth of the pathogen in the presence of EPF (mm). B. bassiana isolates were scored using the modified Bell scale (Bell et al. 1982) based on their ability to suppress mycelial growth of V. dahliae and are presented in Table 2.

Table 2 Scale modified from Bell et al. (1982) used in experiments

Data analysis

Data for this study were performed with the packet statistics program JMP IN (SAS Institute, Cary, NC, 13.0 PC version). Data were examined using ANOVA (one-way analysis of variance). Duncan's multiple range test was used to determine whether there was a significant difference (P ≤ 0.01) among the mean diameters of zones of the EPF inhibition in vitro.

Results

Experiments were performed under laboratory conditions according to the dual culture technique, and the effects of local isolates of B. bassiana on mycelial growth and the mean inhibition (%) of V. dahliae isolates are shown in Table 3. This study had statistically significant (P ≤ 0.01) mycelial growth and inhibition rates of EPF against test pathogens (non-defoliating and defoliating pathotypes) compared to the control Petri plate. Mycelial growths of PHCVd3 (non-defoliating pathotype) and PHCVd47 isolates (defoliating pathotype) of the pathogen were measured as 89.5 and 71 mm in the control Petri dishes (first and second experiment), respectively.

Table 3 Percentage of inhibition of Verticillium dahliae by Beauveria bassiana under in vitro conditions

The lowest mycelial growth against PHCVd3 isolate (non-defoliating pathotype) was in Bb18 (13.2 mm) and Bb1 (15.8 mm) isolates, when B. bassiana isolates were applied, 2 days before V. dahliae, and these isolates were included in the same statistical group. The highest mycelial growth was found with 43.3 mm in BMAUM-M6-4 isolate. The highest percentage of inhibition against the PHCVd3 isolate was determined in isolates Bb18 (85.3%) and Bb1 (82.3%), with a scale value of 1 for these isolates (Fig. 1). The lowest percentage inhibition was detected in the BMAUM-M6-4 isolate at 51.6%. The Bb18 (13.0 mm) and Bb1 (16.0 mm) isolates also showed the least mycelial development relative to the PHCVd47 isolate (defoliating pathotype), and these isolates were included in the same statistical group. The highest mycelial growth was found at 41.7 mm in the BMAUM-M6-4 isolate. The highest percentage of inhibition against the PHCVd47 isolate was determined in isolates Bb18 (81.7%) and Bb1 (77.5%), which received a scale value of 1 (Fig. 1). The lowest inhibition rate was also recorded with the BMAUM-M6-4 isolate (41.3%). When both B. bassiana and V. dahliae were applied simultaneously, the lowest mycelial growth was detected in isolates, Bb1 (17.4 mm) and Bb18 (19.9 mm) and the PHCVd3 isolate and these isolates were included in the same statistical group. The highest mycelial growth was measured as the 45.9 mm BMAUM-M6-4 isolate. The highest percentages inhibition against PHCVd3 isolates were detected at isolates Bb1 (80.6%) and Bb18 (77.8%), which received a scale value of 1 (Fig. 2). The lowest percentage inhibition was observed with the BMAUM-M6-4 isolate at 48.7%. Compared to the PHCVd 47 isolate, the lowest mycelial growth was detected at 17.2 mm for the Bb18 isolate, the highest mycelial growth was detected at 40.6 mm for BMAUM-M6-4, and 38.4 mm for the ET 101 isolate. The highest inhibition rate was 75.8% for the Bb18 isolate, which received a scale value of 1 (Fig. 2). The lowest inhibition rate was 42.8% for the BMAUM-M6-4 isolate (Table 3).

Fig. 1

Antifungal effect of Beauveria bassiana applied to potato dextrose agar plates 2 days before Verticillium dahliae: (a, b) High activity from B. bassiana Bb18 and Bb1 to PHCVd3 (scale value 1) (c, d) High activity from B. bassiana Bb18 and Bb1 to PHCVd47 (scale value 1) (e, f) Control Petri dishes (PHCVd3 and PHCVd47 isolates)

Fig. 2

Antifungal effects of Beauveria bassiana applied concurrently with Verticillium dahliae in potato dextrose agar plates: (a, b) High activity of B. bassiana Bb1 and Bb18 against PHCVd3 (scale value 1) (c) High activity from B. bassiana Bb18 to PHCVd47 (scale value 1) (d, e) Control Petri dishes (PHCVd3 and PHCVd47 isolates)

Discussion

In this study, the highest inhibition rate was recorded in Petri dishes of B. bassiana isolates applied, 2 days before V. dahliae. This may be because B. bassiana possesses mechanisms such as mycoparasites, competition and antibiotics. Researchers have reported that B. bassiana suppresses plant diseases through direct mechanisms such as mycoparasitism, competition and antibiotics and exhibits multiple mechanisms of antagonistic interactions (Ownley et al. 2010).

B. bassiana isolates showed varying degrees of mycelial growth and percent inhibition of both pathotypes. In agreement to our results, Gothandapani et al. (2015) reported that three different entomopatogenic fungi (B. bassiana, M. anisopliae and V. lecanii) were tested against Alternaria porri. All the three EPF showed inhibitory effect against A. porri subjected to in vitro experiments under dual culture technique, conidial germination, mycelia germination and seed germination. The percentage inhibition of mycelial growth of A. porri was 69.24, 56.17 and 45.81%, after B. bassiana, M. anisopliae and V. lecanii treatments, respectively, and the percentage inhibition of conidial germination was detected 97.81, 42.11 and 67.69%, after the same treatments, respectively. B. bassiana exhibited effective antagonism against A. porri, showing the highest percentage inhibition of mycelial growth and conidial growth. As a result, EPF significantly inhibited A. porri. Culebro-Ricaldi et al. (2017) reported a 72% inhibition of pathogen development, when applied to the EPF, B. bassiana 1215 strain, 2 days before F. oxysporum f. sp. lycopersici strain 3. Culture filtrates of B. bassiana SD1, SD7, SD8, SD12, SD14, SD15, and M. anisopliae SD3 at 100% concentration showed varying levels of antifungal activity against B. cinerea, whereas B. bassiana SD8, SD12, SD14, SD15 and M. anisopliae SD3 isolate have been shown to be important in suppressing B. cinerea mycelial growth at low concentrations (Yun et al. 2017). Jaber and Alananbeh (2018) reported that two entomopathogens as endophyte (B. bassiana NATURALIS) and M. brunneum BIPESCO5) found to be antagonistic against three Fusarium species (F. oxysporum, F. culmorum and F. moniliforme). Fuentes et al. (2020) observed that both the EPF, M. brunneum and B. bassiana inhibited mycelial growth of the sunflower wilt pathogens V. dahliae and Cadophora helianthi under in vitro conditions. Deb and Dutta (2021) reported that all 22 native B. bassiana isolates inhibited mycelial growth of the tomato root rot pathogen Pythium myriotylum by 68–82% under in vitro conditions.

Conclusions

It was concluded that Bb18 and Bb1 isolated from B. bassiana isolates included in the study showed a high antagonistic activity against both pathotypes of V. dahliae. Notably, inhibition rates for Bb18 and Bb1 isolates were high in experiments performed, 2 days before the pathogen. The reason for this can be traced back to place competition in B. bassiana and the mechanism of effect of antibiotics. However, plant pathogen control depended not only on B. bassiana isolate characteristics, but also on biotic and abiotic factors. Therefore, this situation should be taken into account in the biological control strategies. In addition, more detailed studies under field conditions are needed on the efficacy of promising isolates, their role in disease management, plant growth promotion and increased yield.