Open Access
Issue
BIO Web Conf.
Volume 18, 2020
IV All-Russian Plant Protection Congress with international participation “Phytosanitary Technologies in Ensuring Independence and Competitiveness of the Agricultural Sector of Russia”
Article Number 00027
Number of page(s) 4
DOI https://doi.org/10.1051/bioconf/20201800027
Published online 06 March 2020

© The Authors, published by EDP Sciences, 2020

Licence Creative Commons
This is an Open Access article distributed under the terms of the Creative Commons Attribution License 4.0, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

1 Introduction

Giant hogweed is one of the most widespread invasive alien species of the Baltic region [1]. The bioherbicides are used as part of complex methods for managing troublesome weeds when the classical methods are insufficient to control their distribution [2]. The isolate of phoma-like fungus Calophoma complanata MF-32.121 is being evaluated as a potential bioherbicide for giant hogweed control [3]. This is a highly specialized plant pathogen that causes the canker of roots, stems and leaves of Apiaceae plant species. It is shown that in Canada C. complanata overwinters in the soil and seed of Pastinaca sativa [4]. The aim of this work is to evaluate an ability of the potential mycoherbicide C. complanata MF 32.121 to overwinter in the conditions of Saint Petersburg area.

2 Materials and methods

The strain of C. complanata MF-32.121 was obtained from the collection of pure cultures of the Mycology and Phytopathology Laboratory of the All-Russian Institute of Plant Protection.

The C. complanata MF-32.121 mycelium was grown on a pearl barley (15 g / 10 ml of water) until concentration 0.7×108 colony-forming units (CFU) per gram. Then, the pearl barley colonized by C. complanata MF-32.121 mycelium was placed on the soil surface, depth 20 cm and at the height of 1.5 m above the ground for wintering in November 2018 at the coordinates of 59.735287°, 30.428694° (Pushkinsky district, Saint Petersburg). Alternatively, it was placed in 4° C refrigerator. After 4 months C. complanata MF-32.121 mycelium was evaluated for viability and pathogenicity.

Plants of Heracleum sosnowskyi at the rosette stage were inoculated with mycelium of C. complanata MF-32.121 (5*104CFU/ml) and incubated for 24 h at 100% relative humidity. By the 14-th day post inoculation, the plants of H. sosnowskyi were placed on the surface of the soil for wintering in November 2018 at the coordinates of 59.735287°, 30.428694° (Pushkinsky district, Saint Petersburg).

Survival of overwintering C. complanata MF-32.121 mycelium was determined by quantity of CFU on potato sucrose agar media. The pathogenicity of reisolates was assessed by the area of H. sosnowskyi leaf necrosis on the 4th day post inoculation (dpi). The H. sosnowskyi plants in the rosette phase was inoculated with an infectious dose of 25 mg / ml (2 ml / plant) at a 24-h period of increased moisture.

Microphotographs of pycnidia and conidia of isolate C. complanata MF-32.121 was obtained using a Leica M165 Stereo Microscope equipped with a digital camera.

Overwintered C. complanata MF-32.121 mycelium was analyzed by the multilocus analysis included amplification and sequencing of a large subunit ribosomal DNA (LSU) and partial beta-tubulin gene were sequenced using primers listed in table 1.

The sugar content in the C. complanata MF-32.121 mycelium was determined using the automated HPTLC system (CAMAG, Muttenz, Switzerland). The TLC plate was developed in 6% boric acid-acetic acid-ethanol-acetone-ethyl acetate (10: 15: 20: 60: 60). Staining was carried out with a resorcinol / sulfuric acid reagent in methanol under heating.

Statistical processing was performed by analysis of variance and when appropriate means comparisons among were tested using an LSD test at p=0.05.

Table 1.

Primers used for PCR

3 Results and discussion

The strain C. complanata MF-32.121 successfully wintered in the Pushkinsky District of Saint Petersburg between November 2018 and February 2019. Similarity original C. complanata MF-32.121 and isolates obtained from the overwintering infectious materials was confirmed by molecular phylogenetic analysis as well by morphological features. Obtained sequences of LSU and beta-tubulin loci of wintered reisolates were compared with those of original C. complanata MF-32.121 (accession numbers of sequences are MH634681 and MH665689.1 for LSU and beta-tubulin gene respectively).

Also, the morphological features of pycnidia, that were formed on the surface of wintered plant material and pearl barley, of the reisolate corresponded to the description of the native strain C. complanata MF-32.121 pycnidia (fig. 1).

The greatest survival of C. complanata MF-32.121 mycelium was observed during wintering on the soil surface (fig. 2). Reisolates from inoculated plants, that were laid on the soil, formed more CFU, than isolates, obtained from overwintered both over and in the soil plants. Also, reisolates from plants, that were wintered on the soil surface, were the most aggressive.

Carbohydrate analysis of overwintered mycelium showed that the highest level of trehalose was observed in the case of the highest viability of pathogen and aggressivity to giant hogweeds (fig. 3). The remaining samples lost viability and pathogenicity by 40-50%, while the content of trehalose was also higher than in the original mycelium (p≤0.05). Trehalose is known to be a major factor in the resistance of microorganisms to drying [2]. However, during wintering, environmental factors also influence the biosynthesis of this disaccharide. Interesting, that the significantly accumulation of trehalose in the Arctic and Antarctic strains of micromycetes growing at low temperatures was shown [8].

Thus, plants inoculated by C. complanata MF-32.121 successfully survives wintering in the conditions of Saint Petersburg area. Pearl barley covered by C. complanata MF-32.121 mycelium successfully survives wintering in the same conditions as well. When favorable conditions have arisen, the C. complanata MF-32.121 mycelium is capable to infecting young giant hogweed. Moreover, it was revealed that a change in the biochemical composition, namely, an increase in the level of trehalose, contributes to the manifestation of a greater tolerance of C. complanata MF-32.121 mycelium to temperature-humidity conditions. In the future, this biochemical indicator can be used as an additional criterion for assessing the quality of mycoherbicides.

This work was supported by RSF grant 16-16-00085.

thumbnail Fig. 1.

A - the native strain C. complanata MF-32.121 pycnidia on the potato sucrose agar; B – the pycnidia observed on the overwintered plants

thumbnail Fig. 2.

Survival of overwintering C. complanata MF-32.121 mycelium LSD0.05=0.5*107. Probe location: 1 - 1.5 m above the ground; 2 - on the soil surface; 3 – 20 cm soil depths.

thumbnail Fig. 3.

The level of trehalose. 1 - pearl barley; 2 - infected by C. complanata MF-32.121 barley before wintering; 3 – infected by C. complanata MF-32.121 barley after wintering at height of 1.5 m above the ground; 4 – infected by C. complanata MF-32.121 barley after wintering on the soil surface; 5 – infected by C. complanata MF-32.121 barley after wintering in 20 cm soil depths. LSD0.05=3*103

References

  • Š. Jahodová, L. Fröberg, P. Pyšek, D. Geltman, S. Trybush, A. Karp, Ecology and management of giant hogweed (Heracleum mantegazzianum) 1–19 (2007) [Google Scholar]
  • A. Berestetskiy, S. Sokornova, Biological Approaches for Controlling Weeds, Publisher: IntechOpen, 63-88 (2018). DOI: 10.5772/intechopen.76936 [Google Scholar]
  • E.L. Gasich, L.B. Khlopunova, A.O. Berestetskiy, S.V. Sokornova, Patent RU 2439141 C1 06.10.2010 (in Russ.) [Google Scholar]
  • R.F. Cerkauskas, Can. J. Plant. Pathol 8(4), 455-458 (1986) [CrossRef] [Google Scholar]
  • S.A. Rehner, G.J. Samuels, Mycol. Res. 98, 625–634 (1994) [Google Scholar]
  • R. Vilgalys, M. Hester, J. Bacteriol. 172, 4238–4246 (1990) [CrossRef] [PubMed] [Google Scholar]
  • K. O’Donnell, E. Cigelnik, Mol. Phylogenet. Evol. 7(1), 103-116 (1997) [Google Scholar]
  • K.V. Sazanova, S.V. Senik, I.Yu. Kirtsideli, A.L. Shavarda, Microbiology (Moscow), 88, 3, 282-291 (2019). [CrossRef] [Google Scholar]

All Tables

Table 1.

Primers used for PCR

All Figures

thumbnail Fig. 1.

A - the native strain C. complanata MF-32.121 pycnidia on the potato sucrose agar; B – the pycnidia observed on the overwintered plants

In the text
thumbnail Fig. 2.

Survival of overwintering C. complanata MF-32.121 mycelium LSD0.05=0.5*107. Probe location: 1 - 1.5 m above the ground; 2 - on the soil surface; 3 – 20 cm soil depths.

In the text
thumbnail Fig. 3.

The level of trehalose. 1 - pearl barley; 2 - infected by C. complanata MF-32.121 barley before wintering; 3 – infected by C. complanata MF-32.121 barley after wintering at height of 1.5 m above the ground; 4 – infected by C. complanata MF-32.121 barley after wintering on the soil surface; 5 – infected by C. complanata MF-32.121 barley after wintering in 20 cm soil depths. LSD0.05=3*103

In the text

Current usage metrics show cumulative count of Article Views (full-text article views including HTML views, PDF and ePub downloads, according to the available data) and Abstracts Views on Vision4Press platform.

Data correspond to usage on the plateform after 2015. The current usage metrics is available 48-96 hours after online publication and is updated daily on week days.

Initial download of the metrics may take a while.