10.6092/issn.2531-7342/15270
Environmental abiotic factors, such as low temperatures, restrict the application of entomopathogenic fungi well-known biological control agents against agricultural pests. Among 60 Iranian isolates collected in Central Iran that were exposed to cold stress, four isolates were identified as potential candidates for further investigation. One hundred percent of the conidia of these four isolates germinated and subsequently produced conidia. Their virulence against Sunn Pest (Eurygaster integriceps Puton) varied between isolates. After applying cold stress (4 °C), Vesh 1-8 isolate with sporulation of 6.5 × 10 7 conidia mL-1, 50% mortality and LT in 3/4 days was the most cold-tolerant isolate. Cold-active germination and growth of the biocontrol fungal isolates are important to industrialize the products with a high potential against target pests in different environmental conditions.
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Italian Journal of Mycology
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Screening and virulence of Iranian isolates of <em>Beauveria bassiana</em> for potential management of Sunn Pest (<em>Eurygaster integriceps</em> Puton) in relation to cold activity
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2023-02-08
2022-07-20
2023-02-08
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Ital J Mycol
Shahram Shahroukhi Khaneghah
2023/02/08
10.6092/issn.2531-7342/15270
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Sunn Pest Research Department, Iranian Research Institute of Plant Protection, Agricultural Research, Education and Extension Organization (AREEO), Tehran, Iran
2531-7342
Iran
en
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White TJ, Bruns T, Lee SJWT, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR protocols: a guide to methods and applications (Innis MA, Gelfand DH, Sninsky J, White TJ, eds). Academic Press, San Diego, CA, pp. 315–322.http://dx.doi.org/10.1016/B978-0-12-372180-8.50042-1
Screening and virulence of Iranian isolates of <em>Beauveria bassiana</em> for potential management of Sunn Pest (<em>Eurygaster integriceps</em> Puton) in relation to cold activity
https://italianmycology.unibo.it/article/download/15270/15631
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                IJM - Italian Journal of Mycology
                ISSN 2531-7342 - Vol. 52 (2023): 17-21
                Journal homepage: https://italianmycology.unibo.it/

Short note

    Screening and virulence of Iranian isolates of Beauveria bassiana
     for potential management of Sunn Pest (Eurygaster integriceps
                    Puton) in relation to cold activity

      Farzaneh Parsi1, Samira Peighami Ashnaei2, Shahram Shahroukhi Khaneghah1
1
  Sunn Pest Research Department, Iranian Research Institute of Plant Protection, Agricultural Research, Education and
Extension Organization (AREEO), Tehran, Iran
2
  Plant Disease Research Department, Iranian Research Institute of Plant Protection, Agricultural Research, Education and
Extension Organization (AREEO), Tehran, Iran

Corresponding author e-mail: farparsi8@gmail.com

ARTICLE INFO
Received 30/08/2022; accepted 28/12/2022
https://doi.org/10.6092/issn.2531-7342/15270

Abstract
Environmental abiotic factors, such as low temperatures, restrict the application of entomopathogenic fungi
well-known biological control agents against agricultural pests. Among 60 Iranian isolates collected in Central
Iran that were exposed to cold stress, four isolates were identified as potential candidates for further investigation.
One hundred percent of the conidia of these four isolates germinated and subsequently produced conidia. Their
virulence against Sunn Pest (Eurygaster integriceps Puton) varied between isolates. After applying cold stress
(4 °C), Vesh 1-8 isolate with sporulation of 6.5 × 107 conidia mL-1, 50% mortality and LT 50 in 3/4 days was the
most cold-tolerant isolate. Cold-active germination and growth of the biocontrol fungal isolates are important
to industrialize the products with a high potential against target pests in different environmental conditions.


Keywords
Beauveria bassiana, cold activity, Sunn Pest, Eurygaster integriceps, Iran


Introduction

Entomopathogenic fungi germinate and infect agricultural pest insects under environmental conditions.
However, environmental abiotic factors can restrict the application of them for biological control.
Considering that low temperatures can affect the virulence of entomopathogenic fungi against pests
as well as their growth and sporulation, cold activity is defined as the ability to grow and germinate
at low temperatures (as low as 5 °C) (Fernandes et al., 2008). The fungus Beauveria bassiana (Bals.)
Vuill is an environmentally safe alternative to chemical pesticides to control agriculturally important
pests. In this study, cold-active germination, growth and conidial production of Iranian B. bassiana
isolates were studied after exposing to cold temperature along with their virulence against Sunn Pest
Eurygaster integriceps Puton (Hemiptera: Scutelleridae).




Department of Agricultural and Food Science in partnership with the Italian Mycological Society (UMI).
The journal is hosted and maintained by ABIS-AlmaDL. © 2023 The Authors. This work is licensed under CC BY 4.0
Parsi et al.                                                     Italian Journal of Mycology 52 (2023): 17-21

https://doi.org/10.6092/issn.2531-7342/15270



Materials and methods

The isolates of B. bassiana were obtained from conidia collected in Sunn Pest cadavers and soil in
the center of Iran including Esfahan, Kerman, Kermanshah, Markazi, Qum, Semnan and Tehran
provinces in 2015-2017 (Parsi, 2019). The conidia were suspended in 10 mL of sterile distilled
water with the addition of 0.02% Tween 80. The conidial suspensions were adjusted to 10 7 conidia
mL-1, and ten µL of conidial suspension was plated on quarter Sabouraud dextrose agar (¼SDA)
medium. The plates were kept at 4 °C and microscopic observations of germination were recorded
after 7 days (Shin et al., 2017). The radial growth (cm) of obtained isolates was measured after 30
days (Davari et al., 2018). Only four out of 60 isolates of B. bassiana which produced conidia at 4
°C were selected in this study. The controls were incubated at 25 °C (no cold stress). The virulence
of the selected four isolates were tested against Sunn Pest E. integriceps. Ten adults in each plastic
lab container were sprayed with 30 mL conidial suspension (107 conidia mL-1) in sterile distilled
water with the addition of 0.03% Tween 80 and the control sprayed only with water containing the
same concentration of Tween 80. Each treatment was replicated three times and repeated on three
separate days.
      Comparison between the cold treatment and control in each isolate was performed with analysis
of Fisher’s least-significant-difference (LSD) test (Webster, 2007), using SAS software. Data were
expressed as means ± standard error (SE) and statistical significance was set at the conventional α <
0.05 level.
      Total DNA of the superior isolate (Vesh 1-8) was extracted, and the internal transcribed
spacer (ITS) region was amplified by PCR using the following universal primers; ITS5 (5′-
GGAAGTAAAAGTCGTAACAAGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′)
(White et al., 1990). PCR products of 600-700 bp were generated corresponding to the ribosomal
DNA (ITS1, ITS2 and 5.8S rRNA). BLAST searches of the GeneBank nucleotide database were
done, and Vesh 1-8 isolate sequence was deposited in the GeneBank (Table 1).

Results and discussion

Out of 60 isolates exposed to the cold stress (4 °C), only four isolates, including Vesh 1-8, N 4-2, V-8
and Virich 3-3 (with 100% germination), produced conidia after 30 days (Figs. 1a,b).
       Although, the conidial production decreased in all four isolates in comparison with the controls
(Fig. 1b); only the conidial production of the isolate Vesh V-8 (4.5×107 conidia mL-1) was not
significantly different from the control (Fig. 1b). On the other hand, the fungal growth rate declined
in all four isolates exposed to the cold stress, and the results showed significant differences compared
with the control (Fig. 1c). Based on the different reduction pattern (Figs 1b,c), it can be concluded that
the responses to the abiotic factors are strain-dependent (Borisade and Magan, 2014). In virulence
assay, the four isolates showed 36-50% accumulated mortality rates with median lethal time (LT 50
day) 3.4-5.2 days, within 14 days (Table 1).
       The isolates Vesh 1-8 and V-8 exhibited the highest virulence against Sunn Pest E. integriceps
with 50% and 40% accumulated mortality rates, respectively (Table 1). Vesh 1-8 isolate with high
mortality (Table 1), germination and conidial production (Figs 1a,b) showed the highest activity after
the treatment at 4 °C.
       Although the selection of entomopathogenic fungi is done based on their virulence against target
pests, survival outside the hosts and sensitivity to abiotic factors are crucial to their efficacy (Lee et
al., 2015; Mossavi and Minassian, 2021). Therefore, field studies need to be conducted to evaluate
their survival and ability to spread in the natural environment (Karimi et al., 2019). Cold activity of
entomopathogenic fungi is as important as their thermotolerance (Parsi and Peighami Ashnaei, 2022),
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               Table 1 - Virulence of the four isolates was evaluated against Sunn Pest (E. integriceps).
               The accumulated mortality rate and median lethal time (LT50 day) were calculated at 4
               days and within 14 days, respectively.

                Beauveria bassiana               Mortality%±SE*         LT50 (d)       Accession Number
                Vesh 1-8                         50.0 ± 0.6ab           3.4            OM760875
                Virich 3-3                       36.6 ± 0.7b            5.2            -
                V-8                              40.0 ± 0.0ab           5.0            -
                N4-2                             36.6 ± 0.7b            4.9            -
                                                              a
                GHA (standard isolate)**         56.6 ± 0.7             3.6            MZ951125
                Tween 80 (0.03%)                 0                      0
               *Values followed by different letters are significantly different (LSD, 5%); **(BotaniGard
               wettable powder, USA from Parker et al., 2003)




               Fig 1. - Conidial germination (a) of four isolates of Beauveria bassiana (Vesh 1-8, N 4-2,
               V-8 and Virich 3-3) after exposure to 4 °C for 7 days; conidial production (b) and growth
               rate (c) of the four isolates after exposure to 4 °C for 30 days; control in 25 °C. Bars on the
               columns indicate standard error. Significant differences * = P < 0.05, ** = P < 0.005.

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to control both the overwintering adults and the summer generation in the crop (Edgington et al.,
2007). Investigating and choosing the native entomopathogenic fungi to survive in a given location
can be most promising to industrialize biological products (Lee et al., 2015). In future studies, the
assessment of the expression of genes related to cold tolerance will highlight the genetic mechanisms
involved in cold activity of the B. bassiana Iranian isolates studied here.

Acknowledgements

We are thankful to Iranian Research Institute of Plant Protection for providing the necessary funds
and facilities to carry out this study.

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