aHcy
`(cid:5)(cid:6)(cid:7)(cid:8)(cid:9)(cid:10)(cid:11)(cid:3)(cid:6)(cid:12)(cid:3)(cid:13)(cid:9)(cid:14)(cid:15)(cid:8)(cid:6)(cid:9)(cid:16)(cid:17)(cid:9)(cid:18)(cid:10)(cid:11)(cid:3)(cid:19)(cid:20)(cid:15)(cid:17)(cid:9)(cid:20)(cid:17)(cid:3)(cid:10)(cid:9)(cid:21)(cid:3)(cid:22)(cid:17)(cid:10)(cid:11)(cid:18)(cid:23)(cid:24)(cid:3)(cid:25)(cid:10)(cid:8)(cid:18)(cid:3)(cid:26)
`see) Journal of Environmental Science and Health, Part B
`& Agricultural Wastes:
`(cid:25)(cid:17)(cid:27)(cid:18)(cid:15)(cid:20)(cid:15)(cid:21)(cid:17)(cid:27)(cid:24)(cid:3)(cid:28)(cid:6)(cid:6)(cid:21)(cid:3)(cid:29)(cid:6)(cid:9)(cid:18)(cid:10)(cid:16)(cid:15)(cid:9)(cid:10)(cid:9)(cid:18)(cid:27)(cid:24)(cid:3)(cid:10)(cid:9)(cid:21)(cid:3)(cid:30)(cid:31)(cid:8)(cid:15)(cid:20)(cid:7)(cid:11)(cid:18)(cid:7)(cid:8)(cid:10)(cid:11)(cid:3)(cid:32)(cid:10)(cid:27)(cid:18)(cid:17)(cid:27)
`Pesticides, Food Contaminants, and Agricultural Wastes
`
` ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/lesb20
`
`
`(cid:33)(cid:19)(cid:19)(cid:34)(cid:35)(cid:3)(cid:36)(cid:25)(cid:8)(cid:15)(cid:9)(cid:18)(cid:37)(cid:3)(cid:36)(cid:38)(cid:9)(cid:11)(cid:15)(cid:9)(cid:17)(cid:37)(cid:3)(cid:5)(cid:6)(cid:7)(cid:8)(cid:9)(cid:10)(cid:11)(cid:3)(cid:23)(cid:6)(cid:16)(cid:17)(cid:39)(cid:10)(cid:31)(cid:17)(cid:35)(cid:3)(cid:23)(cid:18)(cid:18)(cid:39)(cid:27)(cid:35)(cid:40)(cid:40)(cid:41)(cid:41)(cid:41)(cid:42)(cid:18)(cid:10)(cid:9)(cid:21)(cid:12)(cid:6)(cid:9)(cid:11)(cid:15)(cid:9)(cid:17)(cid:42)(cid:20)(cid:6)(cid:16)(cid:40)(cid:11)(cid:6)(cid:15)(cid:40)(cid:11)(cid:17)(cid:27)(cid:43)(cid:44)(cid:45)
`
`Taylor & Francis
`Taylor &FrancisGroup
`
`(cid:19)(cid:17)(cid:9)(cid:27)(cid:15)(cid:18)(cid:15)(cid:14)(cid:15)(cid:18)(cid:46)(cid:3)(cid:6)(cid:12)(cid:3)(cid:5)(cid:6)(cid:7)(cid:8)(cid:9)(cid:10)(cid:11)(cid:12)(cid:6)(cid:13)(cid:14)(cid:3)(cid:27)(cid:18)(cid:8)(cid:10)(cid:15)(cid:9)(cid:27)(cid:3)(cid:12)(cid:8)(cid:6)(cid:16)(cid:3)(cid:17)(cid:21)(cid:15)(cid:43)(cid:11)(cid:17)
`Sensitivity of Trichodermastrains from edible
`(cid:16)(cid:7)(cid:27)(cid:23)(cid:8)(cid:6)(cid:6)(cid:16)(cid:27)(cid:3)(cid:18)(cid:6)(cid:3)(cid:18)(cid:23)(cid:17)(cid:3)(cid:12)(cid:7)(cid:9)(cid:31)(cid:15)(cid:20)(cid:15)(cid:21)(cid:17)(cid:27)(cid:3)(cid:39)(cid:8)(cid:6)(cid:20)(cid:23)(cid:11)(cid:6)(cid:8)(cid:10)(cid:47)(cid:3)(cid:10)(cid:9)(cid:21)
`mushrooms to the fungicides prochloraz and
`(cid:16)(cid:17)(cid:18)(cid:8)(cid:10)(cid:12)(cid:17)(cid:9)(cid:6)(cid:9)(cid:17)
`metrafenone
`
`(cid:5)(cid:17)(cid:11)(cid:17)(cid:9)(cid:10)(cid:3)(cid:48)(cid:7)(cid:49)(cid:6)(cid:14)(cid:15)(cid:50)(cid:24)(cid:3)(cid:19)(cid:14)(cid:17)(cid:18)(cid:11)(cid:10)(cid:9)(cid:10)(cid:3)(cid:51)(cid:15)(cid:11)(cid:15)(cid:52)(cid:10)(cid:53)(cid:17)(cid:14)(cid:15)(cid:50)(cid:4)(cid:51)(cid:10)(cid:8)(cid:54)(cid:15)(cid:50)(cid:24)(cid:3)(cid:48)(cid:55)(cid:8)(cid:56)(cid:9)(cid:18)(cid:3)(cid:22)(cid:10)(cid:18)(cid:14)(cid:10)(cid:9)(cid:15)(cid:24)(cid:3)(cid:48)(cid:56)(cid:27)(cid:47)(cid:11)(cid:55)(cid:3)(cid:57)(cid:8)(cid:17)(cid:21)(cid:15)(cid:20)(cid:27)(cid:24)
`Jelena Lukovié, Svetlana MilijaSevic-Marci¢, Lorant Hatvani, Laszl6 Kredics,
`(cid:30)(cid:18)(cid:18)(cid:15)(cid:11)(cid:10)(cid:3)(cid:19)(cid:47)(cid:58)(cid:20)(cid:27)(cid:24)(cid:3)(cid:29)(cid:27)(cid:10)(cid:43)(cid:10)(cid:3)(cid:59)(cid:56)(cid:31)(cid:14)(cid:60)(cid:11)(cid:31)(cid:46)(cid:15)(cid:24)(cid:3)(cid:34)(cid:10)(cid:18)(cid:10)(cid:53)(cid:10)(cid:3)(cid:61)(cid:7)(cid:21)(cid:7)(cid:49)(cid:24)(cid:3)(cid:33)(cid:14)(cid:10)(cid:9)(cid:10)(cid:3)(cid:59)(cid:15)(cid:20)(cid:6)(cid:3)(cid:62)(cid:3)(cid:33)(cid:14)(cid:10)(cid:9)(cid:10)(cid:3)(cid:25)(cid:6)(cid:18)(cid:6)(cid:54)(cid:9)(cid:15)(cid:49)
`Attila Sztics, Csaba Vagvolgyi, NataSa Duduk, Ivana Vico & lvana Potoénik
`
`(cid:63)(cid:6)(cid:3)(cid:20)(cid:15)(cid:18)(cid:17)(cid:3)(cid:18)(cid:23)(cid:15)(cid:27)(cid:3)(cid:10)(cid:8)(cid:18)(cid:15)(cid:20)(cid:11)(cid:17)(cid:35)(cid:3)(cid:5)(cid:6)(cid:7)(cid:6)(cid:8)(cid:9)(cid:3)(cid:10)(cid:11)(cid:12)(cid:13)(cid:14)(cid:15)(cid:16)(cid:17)(cid:3)(cid:18)(cid:14)(cid:6)(cid:19)(cid:7)(cid:9)(cid:8)(cid:9)(cid:3)(cid:20)(cid:15)(cid:7)(cid:15)(cid:21)(cid:9)(cid:22)(cid:6)(cid:14)(cid:15)(cid:16)(cid:4)(cid:20)(cid:9)(cid:23)(cid:24)(cid:15)(cid:16)(cid:17)(cid:3)(cid:10)(cid:25)(cid:23)(cid:26)(cid:8)(cid:19)(cid:3)(cid:27)(cid:9)(cid:19)(cid:14)(cid:9)(cid:8)(cid:15)(cid:17)(cid:3)(cid:10)(cid:26)(cid:28)(cid:29)(cid:7)(cid:25)
`To cite this article: Jelena Lukovi¢, Svetlana MilijaSevi¢-Marci¢, Lorant Hatvani, Laszl6
`(cid:30)(cid:23)(cid:6)(cid:31)(cid:15)(cid:32)(cid:28)(cid:17)(cid:3)(cid:33)(cid:19)(cid:19)(cid:15)(cid:7)(cid:9)(cid:3)(cid:18)(cid:29)(cid:34)(cid:32)(cid:28)(cid:17)(cid:3)(cid:35)(cid:28)(cid:9)(cid:36)(cid:9)(cid:3)(cid:37)(cid:26)(cid:38)(cid:14)(cid:39)(cid:7)(cid:38)(cid:40)(cid:15)(cid:17)(cid:3)(cid:41)(cid:9)(cid:19)(cid:9)(cid:22)(cid:9)(cid:3)(cid:42)(cid:11)(cid:31)(cid:11)(cid:12)(cid:17)(cid:3)(cid:43)(cid:14)(cid:9)(cid:8)(cid:9)(cid:3)(cid:37)(cid:15)(cid:32)(cid:13)(cid:3)(cid:44)(cid:3)(cid:43)(cid:14)(cid:9)(cid:8)(cid:9)(cid:3)(cid:45)(cid:13)(cid:19)(cid:13)(cid:24)(cid:8)(cid:15)(cid:12)(cid:3)(cid:46)(cid:47)(cid:48)(cid:47)(cid:49)(cid:50)
`Kredics, Attila SzGcs, Csaba Vagvélgyi, NataSa Duduk, Ivana Vico & lvana Potoénik (2021)
`(cid:18)(cid:6)(cid:8)(cid:28)(cid:15)(cid:19)(cid:15)(cid:14)(cid:15)(cid:19)(cid:40)(cid:3)(cid:13)(cid:51)(cid:3)(cid:5)(cid:6)(cid:7)(cid:8)(cid:9)(cid:10)(cid:11)(cid:12)(cid:6)(cid:13)(cid:14)(cid:3)(cid:28)(cid:19)(cid:23)(cid:9)(cid:15)(cid:8)(cid:28)(cid:3)(cid:51)(cid:23)(cid:13)(cid:52)(cid:3)(cid:6)(cid:31)(cid:15)(cid:36)(cid:7)(cid:6)(cid:3)(cid:52)(cid:11)(cid:28)(cid:53)(cid:23)(cid:13)(cid:13)(cid:52)(cid:28)(cid:3)(cid:19)(cid:13)(cid:3)(cid:19)(cid:53)(cid:6)(cid:3)(cid:51)(cid:11)(cid:8)(cid:38)(cid:15)(cid:32)(cid:15)(cid:31)(cid:6)(cid:28)(cid:3)(cid:54)(cid:23)(cid:13)(cid:32)(cid:53)(cid:7)(cid:13)(cid:23)(cid:9)(cid:29)
`Sensitivity of Trichoderma strains from edible mushroomsto the fungicides prochloraz
`(cid:9)(cid:8)(cid:31)(cid:3)(cid:52)(cid:6)(cid:19)(cid:23)(cid:9)(cid:51)(cid:6)(cid:8)(cid:13)(cid:8)(cid:6)(cid:17)(cid:3)(cid:5)(cid:13)(cid:11)(cid:23)(cid:8)(cid:9)(cid:7)(cid:3)(cid:13)(cid:51)(cid:3)(cid:55)(cid:8)(cid:14)(cid:15)(cid:23)(cid:13)(cid:8)(cid:52)(cid:6)(cid:8)(cid:19)(cid:9)(cid:7)(cid:3)(cid:18)(cid:32)(cid:15)(cid:6)(cid:8)(cid:32)(cid:6)(cid:3)(cid:9)(cid:8)(cid:31)(cid:3)(cid:27)(cid:6)(cid:9)(cid:7)(cid:19)(cid:53)(cid:17)(cid:3)(cid:45)(cid:9)(cid:23)(cid:19)(cid:3)(cid:56)(cid:17)(cid:3)(cid:57)(cid:58)(cid:59)(cid:49)(cid:17)(cid:3)(cid:57)(cid:60)(cid:4)(cid:58)(cid:61)(cid:17)(cid:3)(cid:42)(cid:62)(cid:43)(cid:59)
`and metrafenone,Journal of Environmental Science and Health, Part B, 56:1, 54-63, DOI:
`
`10.1080/03601234.2020.1838821
`(cid:49)(cid:48)(cid:63)(cid:49)(cid:48)(cid:64)(cid:48)(cid:65)(cid:48)(cid:61)(cid:58)(cid:48)(cid:49)(cid:47)(cid:61)(cid:60)(cid:63)(cid:47)(cid:48)(cid:47)(cid:48)(cid:63)(cid:49)(cid:64)(cid:61)(cid:64)(cid:64)(cid:47)(cid:49)
`
`(cid:63)(cid:6)(cid:3)(cid:11)(cid:15)(cid:9)(cid:49)(cid:3)(cid:18)(cid:6)(cid:3)(cid:18)(cid:23)(cid:15)(cid:27)(cid:3)(cid:10)(cid:8)(cid:18)(cid:15)(cid:20)(cid:11)(cid:17)(cid:35)(cid:3) (cid:53)(cid:19)(cid:19)(cid:54)(cid:28)(cid:59)(cid:65)(cid:65)(cid:31)(cid:13)(cid:15)(cid:63)(cid:13)(cid:23)(cid:38)(cid:65)(cid:49)(cid:48)(cid:63)(cid:49)(cid:48)(cid:64)(cid:48)(cid:65)(cid:48)(cid:61)(cid:58)(cid:48)(cid:49)(cid:47)(cid:61)(cid:60)(cid:63)(cid:47)(cid:48)(cid:47)(cid:48)(cid:63)(cid:49)(cid:64)(cid:61)(cid:64)(cid:64)(cid:47)(cid:49)
`To link to this article: https://doi.org/10.1080/03601234.2020.1838821
`
`fi Published online: 06 Nov 2020.
`(cid:45)(cid:11)(cid:36)(cid:7)(cid:15)(cid:28)(cid:53)(cid:6)(cid:31)(cid:3)(cid:13)(cid:8)(cid:7)(cid:15)(cid:8)(cid:6)(cid:59)(cid:3)(cid:48)(cid:58)(cid:3)(cid:41)(cid:13)(cid:14)(cid:3)(cid:47)(cid:48)(cid:47)(cid:48)(cid:63)
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`JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH, PART B
`2021, VOL. 56, NO. 1, 54–63
`https://doi.org/10.1080/03601234.2020.1838821
`
`Sensitivity of Trichoderma strains from edible mushrooms to the fungicides
`prochloraz and metrafenone
`
`Jelena Lukovica, Svetlana Milijasevic-Marcica, Lorant Hatvanib, Laszlo Kredicsb, Attila Sz}ucsb, Csaba Vagv€olgyib,
`Natasa Dudukc, Ivana Vicoc, and Ivana Potocnika
`
`aInstitute of Pesticides and Environmental Protection, Belgrade, Serbia; bDepartment of Microbiology, Faculty of Science and Informatics,
`University of Szeged, Szeged, Hungary; cFaculty of Agriculture, University of Belgrade, Belgrade, Serbia
`
`ABSTRACT
`Twenty-two strains of Trichoderma spp. (T. harzianum species complex [THSC], Trichoderma aggres-
`sivum f. europaeum, Trichoderma pleuroti, and Trichoderma pleuroticola) causing green mold dis-
`ease on edible mushrooms (button mushroom, shiitake and oyster mushroom), collected during
`2004–2018 from four countries (Serbia, North Macedonia, Croatia, and Hungary) were examined.
`Based on their ITS (internal transcribed spacer) sequences, strains from shiitake mushroom in
`Serbia were identified as members of the THSC, while in samples obtained from Serbian and
`North-Macedonian oyster mushroom farms THSC, T. pleuroti and T. pleuroticola were detected,
`which represent the first findings in the region.
`In fungicide susceptibility tests, all examined
`Trichoderma strains were found to be highly sensitive to prochloraz (ED50<0.4 mg mL1) and con-
`siderably susceptible to metrafenone (ED50 < 4 mg mL1). The most sensitive taxon to both fungi-
`cides was THSC from oyster mushroom. The toxicity of metrafenone was satisfying and strains
`from oyster mushroom showed the highest sensitivity (ED50 < 1.43 mg mL1), while strains originat-
`ing from button mushroom and shiitake displayed similar susceptibilities (ED50 < 3.64 mg mL1).
`After additional in vivo trials, metrafenone might also be recommended for the control of green
`mold disease in mushroom farms.
`
`ARTICLE HISTORY
`Received 4 September 2020
`Accepted 14 October 2020
`
`KEYWORDS
`Button mushrooms; oyster
`mushroom; shiitake; green
`mold disease; fungicide
`sensitivity test; disease
`control; Trichoderma
`pleuroti; Trichoderma
`pleuroticola;
`Trichoderma harzianum
`
`Introduction
`
`The most commonly cultivated mushrooms worldwide are
`button mushroom (Agaricus bisporus (Lange) Imbach), shii-
`take (Lentinula edodes (Berk.) Pegler) and oyster mushroom
`(Pleurotus ostreatus (Jasq.) P. Kummer). Fungal pathogens
`have a significant negative effect on mushroom yield and
`quality.[1] Green mold caused by Trichoderma spp. is the
`most devastating disease, accounting for mushroom yield
`losses between 60% and 100%.[2,3] The disease is character-
`ized by the presence of white mycelia of fast-growing colo-
`nies that change color into dark green after extensive
`sporulation on the substrate of all three cultivated species.
`Brown necrotic spots and lesions may also appear on mush-
`room fruiting bodies as accompanying symptoms. Serious
`outbreaks appear as substrate areas without mushrooms are
`surrounded by red-pepper mites (Pygmephorus spp.) feeding
`on the pathogenic fungi.[2] Several Trichoderma species were
`identified as aggressive colonizers and predominant causal
`agents of green mold diseases of
`edible mushrooms:
`Trichoderma harzianum sensu lato (T. harzianum species
`complex, THSC), Trichoderma aggressivum f. europaeum
`Samuels & W. Gams and T. aggressivum f. aggressivum
`Samuels & W. Gams on button mushroom; Trichoderma
`pleuroti S.H. Yu & M.S. Park and Trichoderma pleuroticola
`S.H. Yu & M.S. Park on oyster mushroom and THSC on
`
`shiitake mushroom.[4–9] Trichoderma species are cosmopol-
`itan in soils, playing a role primarily in the decomposition
`of wood and other plant materials. However, certain aggres-
`sive species like T. aggressivum and T. pleuroti have been
`found so far only in association with button mushroom and
`oyster mushroom substrate, respectively.[10]
`Disease control usually includes a series of preventive
`measures in mushroom farms: strict hygiene,
`treatments
`with disinfectants and the application of fungicides. Only
`few chemical fungicides are approved, available and officially
`recommended in the mushroom industry,
`including pro-
`chloraz in the European Union, as well as chlorothalonil
`and thiabendazol in North America. [1,11,12] The most effect-
`ive fungicide in mushroom disease control
`is prochloraz,
`which was found to be effective also against the main fungal
`[1,13–17]
`pathogens
`in Serbian mushroom cultivation.
`Nevertheless, decreased sensitivity of Lecanicillium fungicola
`(Preuss) Hassebrauk (dry bubble) and Cladobotryum myco-
`phylum (Oudemans) W. Gams & Hoozemans (cobweb dis-
`ease) to prochloraz was noted in many EU countries.[18–20]
`Recently, metrafenone was introduced to control
`fungal
`pathogens, Cladobotryum spp. and L.
`fungicola in Spain,
`France, Belgium, the UK and the USA.[12,21] However, for
`green mold disease control there is still no alternate option,
`and sensitivity data of Trichoderma strains to fungicides are
`
`CONTACT Ivana Potocnik
`
`ivana.potocnik@pesting.org
`
`Institute of Pesticides and Environmental Protection, Banatska 31b, 11080 Belgrade, Serbia
`
`ß 2020 Taylor & Francis Group, LLC
`
`

`

`scarce. Studies on the efficacy of new fungicides developed
`by agrochemical companies are rare and expensive, espe-
`cially for small crops such as edible mushrooms.[22]
`The objective of this study was the in vitro sensitivity
`testing of different Trichoderma taxa (THSC, T. aggressivum
`f. europaeum, T. pleuroti, and T. pleuroticola), causal agents
`of green mold diseases of cultivated mushrooms (button
`mushroom, shiitake, and oyster mushroom) to the fungi-
`cides prochloraz and metrafenone. Trichoderma strains from
`button mushroom, oyster mushroom and shiitake were
`derived from four neighboring countries (Serbia, North
`Macedonia, Croatia, and Hungary). Furthermore, the study
`presents the results of the first survey and molecular identi-
`fication of oyster mushroom green molds in Serbia and
`North Macedonia, and of shiitake green molds in Serbia.
`Also, strains were characterized based on their morpho-
`logical, pathogenic and ecological features. Strains from but-
`ton mushroom (THSC and T. aggressivum f. europaeum)
`were tested against the fungicide metrafenone only, as their
`identification, virulence and sensitivity testing to prochloraz
`had been performed previously.[17]
`
`Materials and methods
`
`Trichoderma strains
`
`A total set of 22 Trichoderma isolates collected from mush-
`room farms (button mushroom, oyster mushroom and shii-
`take) in four countries, Serbia, North Macedonia, Croatia
`and Hungary, during 2004–2018 (Table 1) was studied.
`Samples of substrate with symptoms resembling green mold
`disease were collected from farms growing oyster mushroom
`in North Macedonia and Serbia and shiitake farms from
`Serbia. Subsequently,
`twelve fungal strains were isolated
`from these samples by taking small pieces (2  2  5 mm),
`immersing them in a 1% sodium hypochlorite solution for
`1 minute, and placing on Potato Dextrose Agar (PDA)
`medium. The plates were incubated for
`four days at
`22 ± 1 C after strain isolation. Six strains from A. bisporus,
`THSC T10, T52, and T54, as well as T. aggressivum f. euro-
`paeum T76, T77 and T85, identified and tested for sensitiv-
`ity to prochloraz in previous studies by Kosanovic et al. [17],
`were used in the current study for sensitivity testing to the
`fungicide metrafenone. The strains have been maintained in
`the culture collection of
`the Institute of Pesticides and
`Environmental
`Protection,
`Belgrade,
`Serbia.
`Four
`Trichoderma strains from Hungary and Croatia, obtained
`from the
`Szeged Microbiology Collection
`(SZMC,
`Department of Microbiology, Faculty of Science
`and
`Informatics, University of Szeged, Hungary), were also
`included in the survey (Table 1).[10,23]
`
`Molecular identification of fungal strains from oyster
`mushroom and shiitake
`
`Species identification of the novel Trichoderma strains from
`oyster mushroom and shiitake farms in Serbia and North
`Macedonia was performed by the PCR amplification and
`
`JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH, PART B
`
`55
`
`sequence analysis of the ITS (internal transcribed spacer) 1
`and 2 regions, as described by Hatvani et al.[24,25] The
`obtained ITS sequences have been submitted to the NCBI
`GenBank (https://www.ncbi.nlm.nih.gov/genbank/; accession
`numbers listed in Table 1).
`
`Virulence test assay of fungal strains from oyster
`mushroom and shiitake
`
`Virulence assay was performed by inoculating the harvested
`pilei of P. ostreatus and L. edodes with conidial suspensions
`(3  106 conidia mL1) prepared from four-day-old cultures
`of all
`tested strains from oyster mushroom and shiitake
`using the method of Bonnen and Hopkins.[26] Stipites were
`removed from all basidiomes and the top of the pilei were
`inoculated with 20 lL of conidial suspension. Three repli-
`cates per each strain were conducted. Sterile water was used
`as a negative control. Inoculated pilei were kept at room
`temperature (22 ± 2 C), and after 2 days, the symptoms were
`rated as follows: 0 ¼ no symptoms; 1 ¼ faint rings around
`the inoculation site; 2 ¼ light brown rings around the inocu-
`lation site; 3 ¼ dark brown rings at
`the inoculation site;
`4 ¼ dark brown rings, some sporulation and some pitting of
`the pileus tissue at the inoculation site; and 5 ¼ symptoms
`extended beyond the inoculation site, severe pitting of the
`tissue, and profuse sporulation.
`
`Morphological studies and growth conditions of fungal
`strains from oyster mushroom and shiitake
`
`Morphological characterization (colony color; conidial shape,
`color and size), growth rate and ecological features of the
`strains were investigated. Growth rate test was performed
`from day 2 to day 6 of cultivation on PDA in darkness at
`22 C. Descriptive statistics of conidial dimensions and
`length/width ratio were made based on 50 measurements
`per
`strain. Mean and range values were reported. An
`Olympus CX41RF microscope (Olympus Life and Material
`Science Europa GMBH, Hamburg, Germany), Moticam 10þ
`digital camera and Motic Images Plus 3.0 software (LLG
`GmbH, Meckenheim, Germany) were used in the study. The
`6-mm agar plugs of each tested strain were placed 1 cm
`from the edge of PDA plate. Radial growth rate of the
`strains was defined in mm h1 by measuring colony diame-
`ters from the edge of mycelial agar plug after 72 h of incuba-
`tion at 22 C and calculated using the Eq. (1):
`
`Radial growth rate mm h1
`ð
`
`Þ
`
`¼ Colony radius mmð
`
`Þ=Incubation period ðhÞ
`
`[17]
`ð1Þ
`
`The effect of temperature on growth was studied on strains
`growing on PDA in darkness at 17, 20, 22, 25, 30, and 35 C
`by measuring colony diameter after 3 days. Four replicates
`per each strain were done.
`
`

`

`56
`
`J. LUKOVIĆ ET AL.
`
`Table 1. Trichoderma strains involved in the study.
`
`Strain code
`T57a
`
`T58
`T59
`T10
`T52
`T54
`T76
`
`T77
`
`T85
`
`SZMC 24039
`(MFBF 10386)
`SZMC 24040
`(MFBF 10387)
`SZMC 12454
`
`SZMC 23033 (A37,
`CPK 2104)
`KG6
`
`KG10
`
`KG13
`
`KM4
`
`KM5
`
`KM12
`
`KM6
`
`KM8
`
`KM11
`
`Species
`
`Origin
`
`Cultivated mushroom
`
`Specimen, year
`of collection
`
`GenBank ITS (reference)
`
`T. harzianum species
`complex (THSC)
`
`Ugrinovci, Serbia
`
`Shiitake
`
`Fruiting body, 2008
`
`MT876593
`
`Button mushroom
`
`Oyster mushroom
`
`Ugrinovci, Serbia
`Ugrinovci, Serbia
`Pozarevac, Serbia
`Zemun, Serbia
`Kula, Serbia
`Lisovici, Barajevo, Serbia
`
`Lisovici, Barajevo, Serbia
`
`Lisovici, Barajevo, Serbia
`
`Croatia
`
`Croatia
`
`Hungary
`
`Hungary
`
`T. aggressivum
`f. europaeum
`
`T. pleuroti
`
`T. pleuroticola
`
`T. pleuroti
`
`T. pleuroticola
`
`T. pleuroticola
`
`Kragujevac, Serbia
`
`T. harzianum species
`complex (THSC)
`
`Kragujevac, Serbia
`
`Kragujevac, Serbia
`
`T. pleuroticola
`
`Kavadarci, N. Macedonia
`
`Kavadarci, N. Macedonia
`
`Kavadarci, N. Macedonia
`
`T. pleuroti
`
`Kavadarci, N. Macedonia
`
`Kavadarci, N. Macedonia
`
`Kavadarci, N. Macedonia
`
`Fruiting body, 2008
`Fruiting body, 2008
`Fruiting body, 2006
`Fruiting body, 2008
`Fruiting body, 2008
`Mushroom
`compost, 2010
`Mushroom
`compost, 2010
`Mushroom
`compost, 2010
`Mushroom
`substrate, 2011
`Mushroom
`substrate, 2011
`Mushroom
`substrate, 2009
`Mushroom
`substrate, 2004
`Mushroom
`substrate, 2018
`Mushroom
`substrate, 2018
`Mushroom
`substrate, 2018
`Mushroom
`substrate, 2018
`Mushroom
`substrate, 2018
`Mushroom
`substrate, 2018
`Mushroom
`substrate, 2018
`Mushroom
`substrate, 2018
`Mushroom
`substrate, 2018
`
`MT876594
`MT876595
`KC555182 [17]
`KC555177 [17]
`KC555183 [17]
`KC555185 [17]
`
`KC555186 [17]
`
`KC555184 [17]
`
`MT876591 [10]
`
`MT876592 [10]
`
`MT876590 [23]
`
`EF392794 [6,24]
`
`MT876581
`
`MT876582
`
`MT876583
`
`MT876584
`
`MT876585
`
`MT876589
`
`MT876586
`
`MT876587
`
`MT876588
`
`aStrains isolated within the frames of the present study are set in bold.
`
`Fungicides
`
`In vitro toxicity of two commercial fungicides: prochloraz
`(MirageVR 45 EC, 450 g L1; Adama Makhteshim Ltd, Beer
`Sheva, Israel) and metrafenone (VivandoVR SC, 500 g L1;
`BASF SE, Ludwigshafen am Rhein, Germany), were tested
`against Trichoderma strains. Six strains from button mush-
`room, THSC T10, T52, and T54 as well as T. aggressivum f.
`europaeum T76, T77, and T85 were tested only against the
`fungicide metrafenone, as their sensitivity to prochloraz was
`previously reported by Kosanovic et al.[17]
`
`In vitro antifungal activity
`
`the selected fungicides against Trichoderma
`Activity of
`strains was tested by macrodilution method on PDA
`
`amended with the tested fungicides, using the preliminary
`concentrations of 0.01, 0.1, 1, 10, 100, and 1000 mg mL 1.
`Based on earlier observations,[17] concentrations of pro-
`chloraz and metrafenone selected for the study included
`0.00243, 0.0081, 0.027, 0.09, and 0.30 mg mL1. The
`selected fungicide concentrations from 1% stock solutions
`were added to sterile molten medium (about 50 C). The
`fungicide-amended media, and the fungicide-free media
`used as control were inoculated with agar disks (Ø 6 mm)
`covered by mycelium taken from the edge of four-day-
`old cultures of Trichoderma strains and incubated at
`20 ± 1 C. Colony diameters were measured after 72 h.
`Mycelial growth in the fungicide-amended media was
`expressed as percentage compared to control using the
`Eq. (2):
`
`Percentage of growth inhibition ¼ 100
`
`
`
`ð
`
`Colony diameter in control plate Colony diameter in tested plate
`Þ=
`Colony diameter in control plate  100
`
` ð2Þ
`
`

`

`Table 2. Colony diameter (mm) of Trichoderma strains on Potato Dextrose Agar at different temperatures, 72 h after inoculation.
`
`JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH, PART B
`
`57
`
`Species
`
`Trichoderma aggressivum
`f. europaeum
`
`Cultivated
`mushroom
`Button mushroom
`
`Trichoderma harzianum
`species complex (THSC)
`
`Colony diameter (mm) Mean ± SE1
`
`Strain
`code
`
`T76
`
`T77
`T85
`T10
`
`17 C
`23.4 ± 3.5e
`
`20 C
`38.6 ± 2.1d
`
`26.0 ± 0.0d
`21.4 ± 3.5e
`24.6 ± 1.8e
`
`44.0 ± 2.7c
`31.6 ± 1.2d
`50.0 ± 2.0d
`
`22 C
`84.5 ± 1.1b
`
`69.5 ± 0.5b
`90.0 ± 0.8b
`88.0 ± 0.0c
`
`25 C
`89.6 ± 0.6a
`
`30 C
`65.4 ± 1.5c
`
`35 C
`3.0 ± 2.1f
`
`97.6 ± 2.1a
`111.4 ± 1.0a
`108.6 ± 2.5b
`
`74.0 ± 6.0b
`56.0 ± 1.7c
`116.4 ± 3.8a
`
`12.0 ± 0.0e
`2.6 ± 0.6f
`1.0 ± 2.1f
`
`Shiitake
`
`Oyster mushroom
`
`Trichoderma pleuroticola
`
`Trichoderma pleuroti
`
`9.0 ± 0.0f
`139.0 ± 0.0a
`86.6 ± 0.6b
`84.0 ± 0.8c
`41.6 ± 0.6d
`24.6 ± 0.6e
`T52
`0.0 ± 3.5e
`81.4 ± 2.3a
`83.4 ± 4.0a
`72.0 ± 0.8b
`56.0 ± 0.6c
`38.6 ± 1.5d
`T54
`55.5 ± 7.8e
`140.0 ± 0.0a
`132.5 ± 1.4b
`107.0 ± 0.6c
`74.0 ± 1.4d
`47.0 ± 0.5f
`T57
`65.0 ± 1.4c
`120.5 ± 1.4a
`124.0 ± 0.0a
`88.0 ± 1.2b
`63.0 ± 2.1c
`33.0 ± 0.7d
`T58
`46.0 ± 3.5d
`128.0 ± 0.0a
`129.0 ± 0.0a
`105.5 ± 1.0b
`78.0 ± 0.7c
`32.5 ± 0.7e
`T59
`64.0 ± 2.8d
`134.0 ± 2.8a
`131.5 ± 0.7a
`102.5 ± 0.5b
`80.0 ± 0.0c
`35.0 ± 0.4e
`KG13
`71.0 ± 2.1d
`138.0 ± 1.4a
`133.5 ± 0.0a
`104.0 ± 1.2b
`80.0 ± 0.0c
`30.5 ± 0.7e
`KG10
`67.0 ± 3.5e
`138.5 ± 2.1a
`133.0 ± 0.7b
`109.5 ± 0.5c
`83.5 ± 1.4d
`46.5 ± 1.4f
`KG6
`28.5 ± 2.1f
`133.0 ± 0.7a
`123.5 ± 0.7b
`97.5 ± 1.5c
`76.5 ± 0.7d
`39.5 ± 0.7e
`KM4
`46.5 ± 3.5e
`140.5 ± 0.7a
`139.5 ± 0.7a
`127.5 ± 0.5b
`100.0 ± 0.0c
`50.0 ± 0.0d
`KM5
`61.0 ± 4.2d
`140.0 ± 0.0a
`140.0 ± 0.0a
`123.5 ± 0.5b
`100.0 ± 0.0c
`48.0 ± 0.7e
`KM12
`25.0 ± 3.5f
`77.5 ± 0.0c
`105.0 ± 0.7a
`87.5 ± 2.5b
`66.5 ± 2.1d
`38.5 ± 2.1e
`SZMC 24040
`53.0 ± 4.2d
`121.5 ± 2.8a
`123.5 ± 1.4a
`102.5 ± 1.5b
`83.0 ± 2.8c
`34.0 ± 3.5e
`SZMC 23033
`62.0 ± 0.0d
`140.0 ± 0.0a
`139.5 ± 0.0a
`123.5 ± 2.1b
`104.5 ± 0.0c
`42.5 ± 0.7e
`KM6
`23.5 ± 0.7f
`82.0 ± 0.7d
`114.0 ± 1.4a
`106.0 ± 0.0b
`90.5 ± 0.0c
`41.0 ± 0.7e
`KM8
`67.5 ± 0.7d
`140.0 ± 0.0a
`139.5 ± 0.0a
`122.5 ± 0.5b
`99.0 ± 0.0c
`49.5 ± 0.0e
`KM11
`69.0 ± 3.5d
`134.5 ± 1.4a
`137.5 ± 2.1a
`110.0 ± 0.0b
`80.0 ± 0.7c
`37.5 ± 0.0e
`SZMC 24039
`63.5 ± 6.3d
`141.0 ± 0.0a
`135.5 ± 0.0a
`119.5 ± 1.0b
`98.5 ± 2.1c
`45.5 ± 0.0e
`SZMC 12454
`1Data are means of four replicates ± SE, standard error of means; a,b,c,d,e,f Strains by same uppercase letter are not significantly different at P < 0.05.
` Kosanovic et al.[17]
`
`F
`
`485.4
`
`244.0
`2013.1
`1171.5
`
`496.3
`569.7
`306.1
`588.3
`943.5
`564.7
`675.1
`790.2
`582.0
`527.2
`1111.6
`197.4
`237.3
`1465.4
`600.1
`4332.0
`550.9
`423.8
`
`Three replicates per strain were used. The experiment was
`repeated twice (assays I and II). Fungicide effects were studied
`by regression analysis (r2>0.95). The estimated effective fungi-
`cide concentrations inhibiting radial mycelial growth by 50%
`(ED50) were determined for each strain by interpolation from
`computer-generated log-probit plots of fungicide concentra-
`tion and relative inhibition. The strains were considered sensi-
`their ED50 values were less than
`tive to a fungicide if
`5 mg mL1, weakly resistant if ED50 was in the range between
`5 and 50 mg mL 1 and resistant if the ED50 value exceeded
`50 mg mL1.[27] A resistance factor (RF) was also calculated
`for each strain according to the Eq. (3):
`
`RF ¼ ED50 of the observed strain
`=ED50 of the most sensitive strain
`
`[28]
`ð3Þ
`
`in the cultivation of shiitake (T57, T58, and T59) were iden-
`tified as members of the THSC (Table 1).
`
`Virulence assay of Trichoderma strains from oyster
`mushroom and shiitake
`
`Five days after inoculation of the harvested pilei of P. ostrea-
`tus and L. edodes, the virulence assay showed that all tested
`Trichoderma strains (THSC, T. pleuroti and T. pleuroticola)
`from oyster mushroom displayed very low virulence levels
`on fruiting bodies (0 or 1 on the scale of 0–5), while all
`tested THSC strains from shiitake displayed high virulence
`levels (5 on the scale of 0–5) (Table 3; Figs. 1 and 2). Sterile
`water used as a negative control produced no symptoms.
`
`According to the RF values, strains were considered to be
`sensitive if the RF was less than 3, weakly resistant if it was
`between 3 and 20, resistant if it ranged between 20 and 80
`and highly resistant if it was over 100.[29]
`
`Results
`
`Molecular identification of Trichoderma strains from
`oyster mushroom and shiitake
`
`The obtained ITS 1 and 2 sequences were analyzed as
`described by Druzhinina et al.[30] Among the strains col-
`lected from the oyster mushroom farm in Serbia, T. pleuroti-
`cola (KG6) and the THSC (KG10, KG13) were detected,
`while T. pleuroticola (KM4, KM5, and KM12) and T. pleu-
`roti (KM6, KM8, and KM11) were recovered from the
`North Macedonian samples. The agents causing green mold
`
`Morphological and growth characteristics of
`Trichoderma strains from oyster mushroom
`and shiitake
`
`During the first three days, all tested strains were character-
`ized by white mycelium, which later became green as the
`result of sporulation. Colony diameter values of all tested
`strains at different temperatures after three days of incuba-
`tion are shown in Table 2. The optimum temperature values
`for the mycelial growth of all tested strains were between 25
`and 30 C. The optimum temperature for THSC strains
`from shiitake, as well as for THSC and T. pleuroti strains
`from oyster mushroom were at 30 C, while it was at 25 C
`for T. pleuroticola strains from oyster mushroom. All strains
`produced green, subglobose, smooth-walled conidia on phia-
`lides after two to five days of incubation on PDA at 20 C.
`Conidial dimensions were 2.13–3.04–3.62  1.88–2.68–4.08 for
`THSC isolates from shiitake; 2.41–3.15–4.41  2.05–2.93–4.53
`
`

`

`58
`
`J. LUKOVIĆ ET AL.
`
`Table 3. Phenotypical and pathogenic characters of Trichoderma strains from cultivated mushrooms.
`
`Cultivated mushroom Strain code
`Button mushroomd T76
`
`Virulencea
`4
`
`Radial growth rate at
`22 C (mm h1) ±SDb
`0.59 ± 0.02
`
`Conidial dimensions (mm)
`(length  width)c
`(2.5)–3.2–(4.0)  (2.2)–2.7–(3.4)
`
`Conidial length/
`width ratioc
`(1.0)–1.2–(1.4)
`
`T77
`T85
`T10
`
`3
`3
`4
`
`0.48 ± 0.01
`0.63 ± 0.01
`0.61 ± 0.00
`
`(2.3)–3.0–(3.6)  (2.0)–2.5–(3.0)
`
`(1.0)–1.2–(1.5)
`
`Species
`
`Trichoderma
`aggressivum
`f. europaeum
`
`Trichoderma harzianum
`species
`complex (THSC)
`
`Shiitake
`
`Oyster mushroom
`
`(2.6)–3.3–(3.6)  (1.9)–2.6–(4.1)
`(2.1)–2.7–(3.6)  (1.9)–2.6–(3.7)
`(2.4)–3.0–(3.6)  (2.0)–2.6–(3.6)
`(2.6)–3.3–(4.4)  (2.1)–3.0–(4.5)
`(2.4)–3.0–(4.0)  (2.2)–2.9–(3.9)
`(1.8)–2.6–(3.2)  (1.8)–2.3–(4.2)
`(1.8)–2.9–(3.9)  (1.7)–2.8–(3.9)
`(2.1)–2.7–(4.2)  (2.1)–2.7–(3.4)
`(2.1)–2.7–(3.6)  (2.0)–2.8–(3.3)
`(2.4)–3.1–(4.0)  (2.1)–2.9–(3.7)
`(2.2)–3.3–(5.4)  (2.4)–3.0–(5.1)
`(1.8)–2.5–(3.3)  (1.8)–2.3–(3.1)
`(2.4)–2.7–(3.5)  (1.9)–2.5–(3.2)
`(2.2)–2.9–(3.5)  (1.7)–2.5–(3.0)
`(2.4)–3.8–(5.5)  (2.6)–3.7–(5.4)
`(2.0)–2.9–(4.7)  (1.9)–2.6–(4.1)
`
`(1.4)–1.1–(0.9)
`(1.1)–1.1–(1.0)
`(1.2)–1.2–(1.1)
`(1.3)–1.1–(1.0)
`(1.1)–1.0–(1.0)
`(1.0)–1.1–(0.8)
`(1.1)–1.0–(1.1)
`(1.0)–1.0–(1.0)
`(1.0)–1.0–(1.1)
`(1.2)–1.1–(1.1)
`(0.9)–1.1–(1.1)
`(1.0)–1.1–(1.1)
`(1.3)–1.1–(1.2)
`(1.3)–1.2–(1.2)
`(0.9)–1.0–(1.0)
`(1.0)–1.1–(1.3)
`
`Trichoderma pleuroticola
`
`Trichoderma pleuroti
`
`0.58 ± 0.01
`3
`T52
`0.50 ± 0.01
`5
`T54
`0.74 ± 0.01
`5
`T57
`0.61 ± 0.02
`5
`T58
`0.73 ± 0.01
`5
`T59
`0.71 ± 0.01
`0
`KG10
`0.72 ± 0.02
`0
`KG13
`0.76 ± 0.01
`1
`KG6
`0.68 ± 0.02
`0
`KM4
`0.88 ± 0.01
`0
`KM5
`0.86 ± 0.01
`0
`KM12
`0.61 ± 0.03
`0
`SZMC 24040
`0.71 ± 0.02
`0
`SZMC 23033
`0.87 ± 0.03
`0
`KM6
`0.74 ± 0.00
`0
`KM8
`0.85 ± 0.01
`0
`KM11
`0.76 ± 0.00
`0
`SZMC 24039
`0.83 ± 0.01
`1
`SZMC 12454
`aVirulence based on a scale of 0–5 with 0 ¼ no symptoms and 5 ¼ severe symptoms;
`bSD, standard deviation;
`cMean and value ranges (min and max) of conidial dimensions and conidial length/width ratio (50 measurements per strain), value ranges (min and max) are
`shown in brackets.
`dKosanovic et al.[17]
`
`Figure 1. Artificial infection of Lentinula edodes fruiting bodies (fifth day); (A)
`control,
`(B) Trichoderma harzianum species
`complex [THSC]
`strain T58
`(5 ¼ profuse sporulation).
`
`1.82–2.86–5.39 
`for THSC from oyster mushroom;
`1.79–2.98–5.36 
`1.72–