The application of the entomopathogenic fungus Akanthomyces muscarius modified GFP to study endophytization

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Entomopathogenic fungi (EPF) of the genus Akanthomyces (formerly Lecanicillium) are one of the most common and important fungal entomopathogens, infecting sucking insects of the order Hemiptera mainly. The fungi can also parasitize on phytopathogenic fungi (rust, powdery mildew). The entomopathogens from these genera reported as endophytes in various plants under natural conditions [1–2], contributing to an increase in plant immunity to pathogens, as well as a decrease in plant colonization by pests. Endophytic colonization of plants by the fungus Akanthomyces lecanii can suppress the growth of the peach aphid [3]. Akanthomyces muscarius strains caused the death of moth when feeding on cabbage colonized by the fungus [4]. Endophytic properties were assessed using the A. muscarius (= Lecanicillium muscarium) strain Vl 72-GFP fluorescently labeled with GFP [5]. The transformation was done by electroporation of germinated conidia of the high-virulent “wild” strain Vl 72 by the pBARGPE1 vector harboring an eGFP gene, showed an expression of fluorescent protein without affecting fungal growth and virulence. The influence of the fungus on the growth rates of beans was revealed when leaves, sterile soil and seeds were treated with a suspension of conidia of 108 spores/ml. On the 7th day, stimulation of the growth of the stems and roots of the beans was observed when the seeds were soaked in a spore suspension of the fungus. When spraying the leaves, only the stem’s elongation was observed. The studied strain colonizes beans irregularly. When treating the seeds, the fungus was isolated in greater quantities from the roots (26%), when spraying the leaves — from the stem (36%), when watering the soil — also from the stem (43%). Infection of A. muscarius plants by spilling the soil was most effective. No effect of endophytization was found on the number of aphids after 14 days of aphid plant colonization. As a result of the introduction of the spores of Vl 72-GFP strain by shedding the soil under flower crops (lantana, gerbera, acanthus) in the greenhouse of Saint Petersburg Botanical Garden, this strain was isolated from the leaves of the Acanthus mollis L. after one month, which confirms the ability of this species to endophytic colonization of plants in greenhouse conditions. Analysis of hyphae Vl 72-GFP in the plant performed on an AxioImager M1 fluorescent microscope demonstrated the same level of fluorescence as in A. muscarius hyphae growing on the media.

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Russian Institute of Plant Protection, Saint Petersburg, Russia

Entomopathogenic fungi (EPF) of the genus Akanthomyces (formerly Lecanicillium) are one of the most common and important fungal entomopathogens, infecting sucking insects of the order Hemiptera mainly. The fungi can also parasitize on phytopathogenic fungi (rust, powdery mildew). The entomopathogens from these genera reported as endophytes in various plants under natural conditions [1–2], contributing to an increase in plant immunity to pathogens, as well as a decrease in plant colonization by pests. Endophytic colonization of plants by the fungus Akanthomyces lecanii can suppress the growth of the peach aphid [3]. Akanthomyces muscarius strains caused the death of moth when feeding on cabbage colonized by the fungus [4]. Endophytic properties were assessed using the A. muscarius (= Lecanicillium muscarium) strain Vl 72-GFP fluorescently labeled with GFP [5]. The transformation was done by electroporation of germinated conidia of the high-virulent “wild” strain Vl 72 by the pBARGPE1 vector harboring an eGFP gene, showed an expression of fluorescent protein without affecting fungal growth and virulence. The influence of the fungus on the growth rates of beans was revealed when leaves, sterile soil and seeds were treated with a suspension of conidia of 108 spores/ml. On the 7th day, stimulation of the growth of the stems and roots of the beans was observed when the seeds were soaked in a spore suspension of the fungus. When spraying the leaves, only the stem’s elongation was observed. The studied strain colonizes beans irregularly. When treating the seeds, the fungus was isolated in greater quantities from the roots (26%), when spraying the leaves — from the stem (36%), when watering the soil — also from the stem (43%). Infection of A. muscarius plants by spilling the soil was most effective. No effect of endophytization was found on the number of aphids after 14 days of aphid plant colonization. As a result of the introduction of the spores of Vl 72-GFP strain by shedding the soil under flower crops (lantana, gerbera, acanthus) in the greenhouse of Saint Petersburg Botanical Garden, this strain was isolated from the leaves of the Acanthus mollis L. after one month, which confirms the ability of this species to endophytic colonization of plants in greenhouse conditions. Analysis of hyphae Vl 72-GFP in the plant performed on an AxioImager M1 fluorescent microscope demonstrated the same level of fluorescence as in A. muscarius hyphae growing on the media.

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Об авторах

Galina Mitina

All-Russian Institute of Plant Protection

Автор, ответственный за переписку.
Email: galmit@rambler.ru
ORCID iD: 0000-0002-7233-2499
SPIN-код: 6185-6527

PhD, Leader Researcher, Laboratory of Microbiological Plant Protection

Россия, Saint Petersburg

Anna Choglokova

All-Russian Institute of Plant Protection

Email: 4oglik@inbox.ru
ORCID iD: 0000-0002-8139-5416
SPIN-код: 4216-1176

Junior Researcher, Laboratory of Microbiological Plant Protection

Россия, Saint Petersburg

Marina Cherepanova

All-Russian Institute of Plant Protection

Email: cherepma@mail.ru
ORCID iD: 0009-0003-2458-7432
SPIN-код: 5469-3244

Research Engineer, Laboratory of microbiological plant protection

Россия, Saint Petersburg

Sergey Timofeev

All-Russian Institute of Plant Protection

Email: ts-bio@ya.ru
ORCID iD: 0000-0001-6664-3971
SPIN-код: 7215-8800

Researcher

Россия, Saint Petersburg

Viacheslav Dolgikh

All-Russian Institute of Plant Protection

Email: dol1slav@yahoo.com
ORCID iD: 0000-0002-2362-2633
SPIN-код: 8462-4319

Employment

Россия, Saint Petersburg

Список литературы

  1. Gуmez-Vidal S, Lopez-Llorca LV, Jansson H-V, et al. Endophytic colonization of date palm (Phoenix dactylifera L.) leaves by entomopathogenic fungi. Micron. 2006;37:624–632. doi: 10.1016/j.micron.2006.02.003
  2. Nicoletti R, Becchimanzi A. Endophytism of Lecanicillium and Akanthomyces. Agriculture. 2020;10(6): 205. doi: 10.3390/agriculture10060205m
  3. Yuningsih D, Anwar R, Wiyono S. Endophytic colonization of entomopathogenic Lecanicillium lecanii (Zimm) Zare & Gams PTN10, and its effect on tobacco resistance against Myzus persicae Sulzer (Hemiptera: Aphididae). IOP Conference Series: Earth and Environmental Science. 2022;974(1):6. doi: 10.1088/1755-1315/974/1/012089
  4. Kuchár M, Glare TR, Hampton JG, et al. Virulence of the plant-associated endophytic fungus Lecanicillium muscarium to diamondback moth larvae. New Zealand Plant Protection. 2019; 72: 253–259. doi: 10.30843/nzpp.2019.72.257
  5. Timofeev S, Tsarev A, Senderskiy I, et al. Efficient transformation of the entomopathogenic fungus Lecanicillium muscarium by electroporation of germinated conidia. Mycoscience. 2019;60(3):197–200. doi: 10.1016/j.myc.2019.02.010

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