Mycoparasitism capability and growth inhibition activity of Clonostachys rosea isolates against fungal pathogens of grapevine trunk diseases suggest potential for biocontrol.

The present study aimed to examine the capability of Clonostachys rosea isolates as a biological control agent against grapevine trunk diseases pathogens. Five C. rosea and 174 pathogenic fungal strains were isolated from grafted grapevines and subjected to in vitro confrontation tests. Efficient antagonism was observed against Eutypa lata and Phaeomoniella chlamydospora while mycoparasitism was observed to the pathogens of Botryosphaeria dothidea and Diaporthe spp. pathogens in in vitro dual culture assays. The conidia production of the C. rosea isolates were also measured on PDA plates. One isolate (19B/1) with high antagonistic capabilities and efficient conidia production was selected for in planta confrontation tests by mixing its conidia with the soil of Cabernet sauvignon grapevine cuttings artificially infected with B. dothidea, E. lata and P. chlamydospora. The length and/or the incidence of necrotic lesions caused by E. lata and P. chlamydospora at the inoculation point were significantly decreased after a three months incubation in the greenhouse on cuttings planted in soils inoculated with the conidia of strain 19B/1, while symptom incidence and severity were unaffected in the case of the pathogen B. dothidea. Based on the above results, we consider C. rosea a promising biological control agent against some grapevine trunk diseases.

Introduction

Grapevine trunk diseases (GTDs) encompass a group of infections caused by fungal pathogens that colonize the woody tissues of grapevines, causing discoloration and necrosis [1] in the lignified vascular tissues. Besides the symptoms observed in the vascular system, these diseases also affect the green parts of the plant: chlorosis and necrosis occurrence on leaves, young shoots deformation, and necrotic spots appearance on the berries. In case of long term infections by Esca disease (one of the most widespread and devastating GTD) the sudden death of the infected part or the whole plant may occur, which phenomenon is called apoplexy [2]. The group of GTDs includes five different syndromes: Black foot disease, caused by Cylindrocarpon spp., Campylocarpon spp. and Ilyonectria spp.; Botryosphaeria dieback, caused by Botryosphaeriaceae spp.; Eutypa dieback caused by Eutypa lata; Petri and Esca disease, caused by Phaeomoniella chlamydospora, Phaeoacremonium minimum and several basidiomycetous fungi like Fomitiporia spp.; Phomopsis dieback caused by Diaporthe spp [3]. The occurrence of GTDs has increased worldwide in the past decades due to the limited use of the efficient preventive and curative techniques. In France, ca. 10% of the productive plants were found to be affected in a 4-year disease incidence estimation of about 700 vineyards [3]. The replacement of dead plants costs around 1.5 billion dollars annually worldwide [4], which is partially due to GTD infections. The highly toxic sodium arsenite has been used to decrease disease frequency, but it is not effective in controlling GTD-related pathogens [5]. Since the ban of this chemical along with other fungicides, there has been no effective way to control GTDs in the European Union. The development of alternative disease management methods, using natural compounds and biological control agents (BCAs) could be a beneficial solution for this problem [1].

To control GTDs, various microorganisms have been tested, including Bacillus subtilis [6], Fusarium lateritium [7] and Pythium oligandrum [8], although Trichoderma species are the most widely examined BCAs in this context. Several studies tested Trichoderma species for the protection of pruning wounds of grapevine against GTD pathogens [9-11] and some studies focused on root or soil application of Trichoderma species [12-14]. Despite the promising results, there are no widely used treatments currently available to protect grafted grapevines from GTD pathogens in nurseries or in the field. Gramaje et al. [15] have emphasized the importance of ecological studies searching for potential BCAs against GTDs in the microbiome of grapevine. One way to obtain a new BCA is to find effective antagonists of pathogens. These organisms should be able to function in the same environmental niches as the pathogen they are expected to control. Therefore, it is reasonable to search for BCAs for a given disease from the microbiota of the host plant. A BCA isolated from the host may control multiple diseases caused by pathogens with similar physiology, epidemiology and ecology [16].

Clonostachys rosea (syn. Gliocladium roseum, teleomorph Bionectria ochroleuca) is a soilborne ascomycetous fungus belonging to the Bionectriaceae family in the Hypocreales order. This species attracts a great attention because of its suitability for biotechnological and pest-control applications [17]. The biotechnological potential of C. rosea covers the biotansformation of several molecules like zearalenon [18], the biodegradation of plastics [19] and the production of biofuels [20]. Because its commercial value, the mass production of C. rosea was also extensively studied both in liquid [21] and solid state [22] fermentations. Similar to several other hypocrealean fungi, C. rosea is known for its antagonistic abilities against numerous plant pathogens, including fungi, nematodes and insects [23, 24]. It can be found globally in soil and decaying plant matter, both in tropical and temperate regions [25] and frequently occurs in grapevine [26-28]. There are only a few studies on the potential use of C. rosea as a biocontrol agent against GTDs [29-31]. The above-mentioned studies investigated a limited number of GTD-related pathogens and all of them lack in planta investigations. The purpose of the present study was to evaluate the biological control capabilities of this fungus against a wide spectrum of GTD-related pathogens, using in vitro and in planta experiments.

Results

Isolation and identification of fungal strains

We carried out a culture-based investigation of the vascular mycobiota of 100 Cabernet sauvignon grapevines. Beside the several GTD-associated fungal species, five isolates of C. rosea were also obtained from plants lacking any internal or external symptoms of GTDs. The morphological characteristics of C. rosea isolates were in accordance with the previous description by Schroers [25]. Colonies growing in the dark on PDA medium at 25°C were whiteish, and showed pale orange coloration when grown under fluorecent light. Conidiophores were Verticillium- or Penicillium- like and produced globose conidia.

The identification of the isolates to the species level was done by sequencing of internal transcribed spacer region and additional loci where necessary. Isolates used in the further investigations are listed in Table 1.

Table 1

List of the isolates used in the present study.

Strain number	Species	Sequenced loci	Grapevine trunk disease
59C/188C/199C/1	Botryosphaeria dothidea	ITS, EF	Botryosphaeria dieback [3]
19B/133C/189C/391C/2100C/1	Clonostachys rosea	ITS	no data
33B/467B/1100B/1	Cadophora luteo-olivacea	ITS	associated with various GTDs [32]
63C/285B/198B/1	Diaporthe ampelina Diaporthe foeniculina Diaporthe fukushii	ITS, EF, ACT	Phomopsis disease [33, 34]
T5/2T14/2T15/2	Eutypa lata	ITS	Eutypa dieback [3]
T17/5T33/152B/1	Phaeoacremonium minimum	ITS, ACT	Petri disease, Esca [3]
23A/537A/348C/4	Phaeomoniella chlamydospora	ITS

ITS: internal transcribed spacer; EF: partial transcription elongation factor 1-α gene; ACT: γ-actin gene

In vitro confrontation tests of C. rosea isolates with GTD-related fungi

Growth inhibitions of C. rosea strains against the GTD-related pathogens were observed in in vitro confrontation tests and the measured percental inhibition rates are summarized in Table 2. The experiments were conducted by culturing the GTD pathogens on PDA plates in the presence or absence of the C. rosea isoaltes and measuring their growth rate.

Table 2

Growth inhibition of C. rosea isolates against GTD pathogens.

GTD pathogens	Clonostachys rosea isolates
	19B/1	33C/1	89C/3	91C/2	100C/1
Botryosphaeria dothidea	9.7 ± 9.7	8.8 ± 6.4	1.9 ± 1.6	-1.0 ± 5.0	12.9 ± 21.3
Cadophora luteo-olivacea	20.3 ± 15.7	8.1 ± 7.5	7.9 ±7.3	7.1 ± 9.4	12.6 ± 14.7
Diaporthe ampelina	3.0 ± 1.4	2.0 ± 2.8	-10.0 ± 2.8	-6.0 ± 2.8	16.0 ± 5.7
Diaporthe foeniculina	13.75 ± 8.8	22.5 ± 3.5	22.5 ± 3.5	18.8 ± 5.3	18.8 ± 1.8
Diaporthe fukushii	-14.0 ± 8.5	-14.0 ± 2.8	-22.0 ± 2.8	-18.0 ± 2.8	-18.0 ± 2.8
Eutypa lata	24.8 ± 7.0ab	24.8 ± 1.4ab	17.8 ± 2.2a	19.2 ± 3.8ab	29.0 ± 1.7b
Phaeoacremonium minimum	-14.5 ± 8.2a	3.7 ± 12.6ab	11.4 ± 14.0ab	-14.5 ± 13.5a	29.8 ± 0.9b
Phaeomoniella chlamydospora	38.0 ± 5.6ab	28.8 ± 9.4a	47.1 ± 2.8b	43.1 ± 9.4ab	54.0 ± 2.0b

Mean values of the growth inhibition rates (RGI %) of GTD-related pathogens by C. rosea isolates in dual cultures ± standard deviations. Significantly (p< 0.05) differing values of different C. rosea strains measured -by Tukey’s HSD test- in the case of the same pathogen were indicated as significance groups.

High RGI % values were measured in case of E. lata (17.8–29 RGI%) and P. chlamydospora (28.8–54 RGI%) species with all the tested C. rosea strains. Efficient growth inhibition of these pathogens was accomplished even before the fungal colonies could establish physical contact with C. rosea strains (Fig 1A and 1B) indicating the antagonistic activity of the BCA on them. Very low, or no growth inhibition was observed in the case of B. dothidea, Cadophora luteo-olivacea, and P. minimum pathogens (-14.5–29 RGI%). Interestingly, the three Diaporthe species tested showed varying responses to in vitro confrontation with C. rosea. For example, D. ampelina mostly showed no change in growth rate, D. foeniculina exhibited weak growth inhibition (18.8–22.5 RGI%) with all C. rosea strains, while the growth rate of the D. fukushii isolate showed positive change with all C. rosea strains (-14 to -22 RGI%). This latter is an unexpected result and we consider it worthy of further research, though we could not pursue this direction for this particular paper. We observed overgrowth of C. rosea mycelia on the colonies of confrontation partners after a long incubation in case of B. dothidea (Fig 1C) and Diaporthe spp. (Fig 1D).

Fig 1

In vitro confrontation tests of C. rosea 19B/1 strain with GTD pathogens growing on potato dextrose agar medium.

(a) P. chlamydospora, 15 days post inoculation (dpi); (b) E. lata, 8 dpi; (c) B. dothidea, 15 dpi; (d) D. ampelina, 15 dpi.

Overgrowth rates were measured and summarized in Table 3. The highest values were measured in the case of B. dothidea (16.2–18.8 mm) and there was also notable overgrowth above the Diaporthe spp. colonies (6.5–18.5 mm). There were no significant differences between the C. rosea strains in case of both pathogen taxa.

Table 3

Mycoparasitism of GTD pathogen colonies by C. rosea isolates.

	Clonostachys rosea isolates
GTD pathogens	19B/1	33C/1	89C/3	91C/2	100C/1
Botryosphaeria dothidea	17.2 ± 1.9	18.8 ± 0.8	17.3 ± 2.5	16.5 ± 2.6	16.2 ± 2.8
Diaporthe ampelina	6.5 ± 0.5	11.5 ± 0.7	12.0 ± 2.8	15.5 ± 2.1	13.3 ± 0.4
Diaporthe foeniculina	17.2 ± 1.91	18.0 ± 1.4	10.5 ± 0.7	16.0 ± 1.4	18.5 ± 0.7
Diaporthe fukushii	10.5 ± 0.7	10.5 ± 0.7	5.5 ± 0.8	12.5 ± 0.7	14.3 ± 1.0

Mean overgrowth of the C. rosea strains (mm) above the colonies of some GTD-related pathogens ± standard deviations. The overgrowth was measured 19 days post-inoculation. Significances of differences between the C. rosea isolates were determined by Tukey’s HSD test, resulting in no significant differences between them.

The ability of the tested C. rosea strains to colonize some GTD-related pathogens in the dual cultures suggests mycoparasitism of the affected pathogens. This phenomenon was visually confirmed by microscopic examinations in the case of B. dothidea and Diaporthe spp. isolates (Fig 2) co-cultured with C. rosea isolates on cellophane water agar with a subsequent stainig of viable cells with tetrazolium dye. The tested C. rosea strains showed intracellular growth both in Diaporthe spp. (Fig 2A) and B. dothidea (Fig 2B). Coiling of C. rosea around the hyphae of these plant pathogens was also observed (Fig 2C).

Fig 2

Microscopic examination of confrontation zones between C. rosea 100C/1 strain and GTD pathogens.

Parasitism of C. rosea on D. ampelina (a) and B. dothidea (b,c) hosts after staining with 5 mM MTT for one hour. Arrows mark C. rosea mycelia. Scalebars represent 10 μm.

Conidia production of C. rosea isolates

The conidia production of the five C. rosea strains were measured on colonies growing under artificial light, on PDA plates and summarized in Fig 3. The most efficient conidia producer was 33C/1 followed by the 19B/1 strain, with no significant difference in the conidia production of these two strains.

Fig 3

Comparison of conidia production by C. rosea isolates.

Isolates were grown on potato dextrose agar medium for six days under fluorescent light, at room temperature. The numbers of produced conidia were normalized to colony surface.

In planta confrontation tests of C. rosea 19B/1 isolate with GTD pathogens

The effects of C. rosea on the development of GTD symptoms were examined on one year old cuttings. Plants were wounded and inoculated in the xylem by the growing mycelia of B. dothidea, E. lata or P. chlamydospora and grown in a greenhouse in an untreated medium or in soil amended with the conidia of C. rosea 19B/1 isolate. Necrotic lesions can be observed in the grapevine cuttings inoculated with GTD-related pathogenic fungi (Fig 4A), while mock-inoculated plants did not show this symptom. Necrotic regions developed deep in the woody tissues in case of B. dothidea and E. lata, while the tested P. chlamydospora isolate necrotized the woody tissues just under the bark of the cuttings (Fig 4A). The length of the necrotic lesions caused by E. lata and P. chlamydospora were significantly decreased in case of cuttings planted in C. rosea-amended soil, while the disease severity was unaffected by the BCA in case of plants infected with B. dothidea (Fig 4B). The re-isolation of C. rosea 19B/1 from cuttings not inoculated with a pathogen showed a declining trend from bottom to top. From the base of the cuttings, the strain was re-isolated from three out of five plants, only one out of five cases at the center of the internode, and it was not found in any of the wound tissue samples. The colony forming unit (CFU) number of the 19B/1 strain increased from 104 to 105 CFU/g in the soil after 90 days of incubation in a greenhouse. It was also notable, that growing mock inoculated cuttings in C. rosea-amended medium did not result in any damage on the plants.

Fig 4

Effects of C. rosea 19/B1 isolate on the development of vascular necrosis caused by GTD pathogens.

(a) Representative photographs of wood necrosis developed on Cabernet sauvignon cuttings mock inoculated or infected with B. dothidea, E. lata and P. chlamydospora. Cuttings were grown for 90 days in greenhouse in soil with (upper row) or without (bottom row) 104/g conidia of C. rosea 19/B1 strain. Scale bars represent 1 cm. (b) Mean lengths and standard deviances of necrotic lesions developed on infected cuttings grown in the untreated or C.rosea-amended soils. Numbers above the columns represent the number of symptomatic plants out of five infected cuttings. Asterisks mark significance of differences (* p<0.05, ** p<0.005).

Discussion

Our study demonstrates that C. rosea is a potent antagonist of various pathogens causing GTDs, based on the antibiotic and mycoparasitic capabilities of this fungus. Furthermore, the results of the in planta confrontation tests suggest, that C. rosea can be effectively applied in the soil to prevent the development of GTD-related symptoms on grapevine.

The strains of C. rosea tested exhibited particularly strong growth inhibition against P. chlamydospora and E. lata species. The inhibition was visible even before C. rosea established physical contact with the pathogen. This suggests the secretion of antibiotic compounds by the C. rosea isolates. Antagonism of C. rosea against P. chlamydospora was previously shown by Silva-Valderrama et al. [31] however antibiosis was not mentioned in that study. Our study is the first report on the antagonistic activity of C. rosea against E. lata. One possible explanation of this phenomenon could be the previously demonstrated production of antifungal compounds by C. rosea [35]. Contrary to the findings of Silva-Valderrama et al. [31] on the antibiotic activity of C. rosea against the botryosphaeriaceous species Diplodia seriata and N. parvum our results did not indicate this phenomenon in the case of B. dothidea. This may be explained by the physiological polymorphisms of both the pathogenic taxa and C. rosea.

Besides the growth inhibition of P. chlamydospora and E. lata, the observed mycoparasitic behavior of the C. rosea isolates on B. dothidea and Diaporthe spp., as inferred from the intracellular growth and coiling of C. rosea hyphae around pathogen hyphae, may suggest different antagonistic strategies of C. rosea against different pathogens. The fact, that only the C. rosea cells were stained by the MTT viability dye, suggests the necrotrophic nature of the parasitism. These results are in accordance with previous studies on the mycoparasitism of C. rosea on several phytopathogenic fungi, including Botrytis cinerea [36], Fusarium oxysporum [37], Sclerotinia sclreotiorum [38], or even Trichoderma spp. [39]. While the mycoparasitic behavior of C. rosea is well known and also demonstrated on the GTD-related pathogens D. seriata and N. parvum [31] our study is the first report of this phenomenon in the case of Diaporthe spp.

The observed differences in conidia production among the tested C. rosea strains may have implications for their potential use as BCA. The intense sporulation of a fungus used as a BCA is advantageous, because generally conidia are used as inoculum for the treatment of plants, higher sporulation leads to more cost-effective production of a BCA. Efficient spore-producing strains are also likely to be more persistent and intensively distributed in the treated plantations. It is also important, that the compounds responsible for the antibiotic activity of a BCA fungus are usually secondary metabolites. These molecules generally are produced during the stationary phase of fungal growth, alongside with the formation of conidia [40].

Because the causal agents of GTDs are colonizing the vascular tissues of grapevine, the applicability of any BCA against them is strongly dependent on its ability to grow in the tissues of the host plant. The external and internal colonization of host by C. rosea was demonstrated previously on cucumber [41] and this fungus was also reported in the tissues of grapevine [26-28]. Our results on the re-isolation of C. rosea from the soil and grapevine cuttings showed differences according to the sampling point. The 19B/1 isolate established successfully in the soil and was also able to increase its cell number by about ten-fold. The strain efficiently colonized the woody tissues at the base of the cuttings but was rarely re-isolated from the upper parts of the plants. These results may partly explain the different biocontrol efficacy of 19B/1 strain against the different GTD-related pathogens, observed in case of the in planta experiments. The C. rosea significantly inhibited the necrosis development in case of E. lata and P. chlamydospora, which are susceptible to the antibiotic effects of C. rosea. This suggests that the antibiotic compounds are secreted by C. rosea in the soil, or in the vascular tissues at the base of the cuttings and transported by the xylem sap to the inoculated pathogens. Another possible mode of action which can result in the inhibition of pathogen growth in the absence of direct contact is the triggering of plant defense mechanism by the biocontrol agent. This phenomenon was previously demonstrated in the case of C. rosea for example against B. cinerea infection tomato [42]. The fact that C. rosea was not able to colonize the upper parts of the cuttings explain its ineffectiveness against B. dothidea which species is not susceptible to the antibiotic effect of C. rosea, but susceptible to mycoparasitism. The latter mode of antagonism requires the establishment of physical contact between the parasite and the prey, which could not be realized in the time period of the experiment due to the relatively slow growth of C. rosea in grapevine cuttings. However, field application of C. rosea may result in efficient protection against B. dothidea, allowing a longer time period for a near-systematic colonization of grapevine woody tissues by C. rosea.

Overall, the above results suggest that C. rosea has potential as a potent BCA against a wide range of important fungal pathogens of GTDs. The similar results observed in the in planta and in vitro growth experiments suggest that, in practice, antifungal compounds produced by C. rosea may be more important for effective biocontrol purposes than mycoparasitism per se, although even the latter could be a promising long-term strategy if near-systemic colonization of grapevine trunks by C. rosea can be achieved.

Materials and methods

Isolation and identification of fungi from grafted grapevines

One hundred Cabernet sauvignon clone VCR8 grafted on SO4 rootstock were used for the isolation of endophytic fungi. The one year old plants were obtained from an Italian nursery in 2018, shipped bare-rooted, and processed immediately after arrival. The grapevines were symptomless and guarantied to be virus-free. Discs were cut from the graft union, two cm below the graft union, and at the base of the grapevines. The discs were surface sterilized by placing the discs in 70%v/v ethanol, sodium hypochlorite (4%m/v available chlorine), and again in 70%v/v ethanol for two minutes each. The sterilized discs were cut to pieces and placed on potato dextrose agar (PDA) medium amended with 10 μg/ml oxytetracycline to prevent bacterial growth. Plates were incubated at 25°C in the dark. Small portions of emerging mycelia were subcultured to obtain monoclonal isolates. Fungi were identified based on morphological characteristics and by PCR amplification and sequencing of the internal transcribed spacer using ITS1F [43] and ITS4 [44] primers. Where necessary, additional genes were sequenced for unambiguous identification: partial transcription elongation factor 1-α gene with EF-728F and EF-986R primers [45] and/or partial γ-actin gene using ACT-512F and ACT-783R primers [46].

In vitro confrontation tests

To investigate the antagonistic ability of the C. rosea strains, confrontation tests were carried out against GTD pathogens. The pathogenic isolates were inoculated individually and in dual cultures with the C. rosea strains at five cm distance on PDA medium. For the inoculations 3 mm discs were cut from the edge of fungal colonies growing on PDA medium. The plates were kept in the dark at 25°C. When the pathogen colony approached the C. rosea colony to a distance of 5 mm, which happened after 4 to 14 days of incubation, depending on the fastest-growing isolate of the species in question, colony diameter was measured for all cultures of that species and radial growth inhibitions (RGI%) were calculated as described elsewhere [38]. Where observed, the overgrowth of C. rosea isolates on the colonies of pathogenic fungi was measured. All experiments were done in triplicates.

Microscopic investigation of mycoparasitism

In order to visually inspect the mycoparasitism of C. rosea on Botryosphaeria dothidea and Diaporthe spp. (the species where overgrowth of C. rosea was observed in confrontation tests), these pathogenic fungi were inoculated in dual cultures with C. rosea strains on 2% water agar covered with a piece of sterile cellophane. For the inoculations 3 mm discs were cut from the edge of fungal colonies growing on PDA medium. The plates were incubated at 25°C until physical contact was established between the growing colonies. Confrontation zones were stained with 5 mM MTT (thiazolyl tetrazolium bromide) solution for one hour at 25°C. Stained sections were cut from the cellophane, placed on microscope slides and examined with Alpha BIO-5f (Optika, Italy) microscope, equipped with Artcam-500MI (Artray, United Kingdom) digital camera.

Quantification of sporulation of C. rosea

The conidia production of C. rosea isolates were measured by a method described previously [47] with some modification. The tested isolates were mass inoculated on PDA plates with conidial suspensions, in three replicates. The plates were incubated under fluorescent light for eight days at room temperature (21±2°C). After the incubation, six-mm wide agar plugs were cut from the center of the colonies and suspended in one ml of 0.01%v/v TWEEN 80 solution. The number of the spores was determined with Bürker-chamber. Total conidia numbers were normalized to the colony surface.

In planta confrontation tests

Cabernet sauvignon cuttings were wounded using a drill and inoculated with mycelial plugs (dia. 3 mm) of three GTD-associated pathogen species growing on PDA medium at 25°C for one week, while sterile agar plugs were placed in the wounds as control. One isolate of E. lata (T15/2), B. dothidea (99C/1) and P. chlamydospora (48C/4) each was used for artificial infections. Two of three pathogenic species (E. lata, P. chlamydospora) were chosen because they exhibited strong growth inhibition by C. rosea and the third (B. dothidea) represented a fungus that was highly susceptible to mycoparasitism by C. rosea, but showed no growth inhibition. C. rosea 19B/1 strain was used for in planta confrontation tests. One set of cuttings were planted into soil inoculated with 104 conidia/g of the 19B/1 strain and another set of the cuttings were planted into soil not inoculated with C. rosea. Soil was inoculated by mixing with conidial suspension obtained from 10 PDA plates prepared as described in the previous section. Five cuttings were used for each soil × pathogen combination. Plants were incubated in greenhouse with ambient light conditions. Partial control of temperature was achieved by automatically opening top windows activated at elevated temperatures. After 90 days of incubation (from June to August in 2020) the cuttings were uprooted, cut longitudinally after removing the bark, and the length of necrotic lesions was measured. The re-isolation of the C. rosea strain 19B/1 was carried out from the inoculated soil and also from three different points from the mock-inoculated cuttings (base, center of internode, wound) grown in the inoculated soil. Serially diluted suspensions of soil were prepared in sterile distilled water and streaked on PDA plates. Re-isolation of fungi from cuttings was done as described above in case of grafted grapevines. Fungi were grown at 25°C temperature in the dark. The colonies of C. rosea were selected according to morphological characteristics and their identity was validated by sequencing the ITS region.

Statistical analysis

Statistical comparisons were made by GraphPad Prism 5 software demo version (GraphPad Software, San Diego California USA, www.graphpad.com) using Tukey’s HSD in case of in vitro confrontation tests, one-way ANOVA in case of conidia production measurement and student t-test in case of in planta conformation tests. Diagrams were generated with the same software and the layout was edited by Adobe Photoshop CS5 demo version.

7 Jul 2022

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Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Manuscript entitled “Mycoparasitic and growth inhibition capabilities of Clonostachys rosea isolates against grapevine trunk diseases fungal pathogens suggest potential for biocontrol” mainly talked about C. rosea isolates exhibited promising biocontrol ability against some grapevine trunk diseases. The manuscript is well written, and the results provide useful information for expanding the pathogen range by C. rosea biocontrol. Some points need to be addressed before it can be publication.

1. Line 63-69. Comprehensive review articles should be cited for introducing Clonostachys rosea. e.g. Sun et al. 2020 “Biology and application of Clonostachys rosea”.

2. Line 75 and 232. Identification of fungal strains needs combined morphological characteristics and molecular sequencing.

In line 232, the author declared that the identification of fungal strains would depend on morphological and molecular method. However, in Line 75, the results were mainly focus on ITS amplication. Then what about the morphological characteristics of these fungi? Especially the five C. rosea stains, are their morphological characteristics were same to known C. rosea strains?

3. Table 2. Why the significance analysis was only used for pathogens of Eutypa lata, Phaeoacremonium minimum and Phaeomoniella chlamydospore, but not for other pathogens in this Table?

4. Line 98. Changed “antibiotic” to “antagonistic”.

5. Line 105. Overgrow is a very important phenomenon for mycoparasitism. In this study, the author found the growth rate of Diaporthe strains were nearly not affect by C. rosea. Therefore, how about the overgrow behavior for C. rosea and Diaporthe strains? Just like in Fig.1 c and d, both two strains were overgrowth each other, or only C. rosea overgrowth pathogen strains? From Fig.1, it same like both the two strains are overgrowth each other.

Dose the single inoculation of C. rosea and pathogen strains were set as control in this study?

6. Figure 1, why only (b) E. lata was investigated at 8 dpi, but other pathogens were at 15 dpi?

7. Line 169-172. The sentence needs rewrote as “Our report is the first on the antagonistic activity of C. rosea against E. lata. Contrary to the findings of Silva-Valderrama…”

Confrontation test in plate could reflect the antagonistic activity of C. rosea against pathogens. However, antagonism had several mechanisms, and produce antibiotic is only one of the mechanisms. Therefore, from the confrontation test result, the author could provide conclusion of C. rosea had antagonistic activity against E. lata, but not antibiotic activity. Also the next sentence, C. rosea could produce many compounds with fungicidal activity, is not appropriate.

Reviewer #2: This paper described a study of determining the effectiveness of Clonostachys rosea in controlling fungal pathogens causing grapevine trunk diseases (GTDs). The authors observed significant pathogen growth inhibition in both in vitro and in planta conditions. The reported findings contribute to the collective knowledge of biocontrol agents of C. rosea, especially with regard to in planta conditions. However, the following areas should be mentioned to improve the manuscript:

1. In abstract, experimental data should be provided to support the results and conclusion.

2. The authors identified eight pathogenic species of grapevine trunk diseases basing on the previous researches, therefore, the related references should be supplemented in the Table. Meanwhile, C. rosea strains were also isolated from the plants. We cannot regard them as beneficial microorganisms before a primary test of pathogenicity to grapevine is performed, especially some C. rosea isolates were noticed to significantly promote the growth of some GTD pathogens in vitro test (Table 2).

3. The authors should provide the context when presented the results. It is hard to fully understand the terms used in the results section, however, a short description of the study method and arrangement will help the readers to understand the data presented. Also, the results of Table 2 and Table 3 need to be clearly described in the text.

4. The authors thought the in planta and in vitro experiments suggest “in practice, antifungal compounds produced by C. rosea may be more important for effective biocontrol purposes than mycoparasitism”. It seems arbitrary, for the fungus was mainly found colonizing in soil and the base of the grapevine. In that case, induced resistance to the diseases is more involved, which has been verified in previous studies. It should be mentioned in discussion.

5. Standard three-line tables should be used in the manuscript with the SI units in the tables. The notes should be put below the table, separating with the title.

6. Supplement statistical analysis in the tables.

7. The figures should be improved. Fig. 1, which C. rosea isolate(s) was used? It should be marked. Fig. 2, clarify the arrows and change the bars to a line instead of an area. Fig.3, Rewrite the title to show the research content and put “�  106” in the title of Y-axis. Different colors for the five isolates are not necessary.

8. Necrotic lesions were presented when inoculated with the pathogens (Fig.4), and the authors also mentioned that “while mock-inoculated plants did not show this symptom”. Here, an uninfected cutting should be presented together with the infected cuttings in the figure.

9. In discussion, the authors thought “only the C. rosea cells were stained by the MTT viability dye, suggests the necrotrophic nature of the parasitism”, and “lack of specific structures”. However, different infection structures have been observed in some mycoparasites, including C. rosea.

10. The authors should provide more information about the grapevine samples where the isolates were seperated, for example, the geographical location, cropping years, infected or not, status of soil.

11. In the section of methods, the experimental procedure should be described with more details, therefore, the readers can clearly follow your steps.

12. The term of “sporulation rate” is not correct.

13. The full names of genus should be used when first appear, then only abbreviations are required if not confused.

Furthermore, the authors should consider the following grammatical revisions:

L2-4. I would suggest to change the title to “Mycoparasitism capability and growth inhibition activity of Clonostachys rosea isolates against fungal pathogens of grapevine trunk diseases suggest potential for biocontrol”.

L14. Write the exact number of the pathogens instead of “several”.

L17-18. Change to “to the pathogens of Botryosphaeria dothidea and Diaporthe spp.”.

L19. Change “on in vitro grown colonies” to “on PDA plates”.

L21-22. “by inoculating… into the growth medium” is not consistent with the method.

L31-32. Can be revised to “chlorosis and necrosis occurrence on leaves, young shoots deformation, and necrotic spots appearance on the berries.”

L33-34. There are two mis-spelt words “widespred” and “occour”, and the sentence should be revised to “… the sudden death of the infected part or the whole plant may occur…”.

L47. Change “pest management” to “disease management”.

L81. In Table 1 use “strain number” instead of “strain ID”.

L86. It should be “Growth inhibitions of C. rosea strains …”.

L90. Change “percental radial growth inhibitions” to “growth inhibition rates”.

L115. Change to “Mycoparasitism of …”

L141. Cancel “were”.

L143. Change “developed by” to “caused by”.

L151. Change to “the development of vascular necrosis caused by…”.

L167. Change “This indicates…” to “This suggests…”.

L169-170. Change to “Our study is the first report…” .

L191. Cancel the word “therefore”.

L199. Change to “this fungus was also reported in the tissues of grapevine”.

L207. Cancel the word “species”.

L211. Change to “but susceptible to mycoparasitism”.

L218: Cancel the comma after the word “suggest”.

L251-252. Change “on cellophaned water agar (2 %m/v)” to “on 2% water agar covered with a piece of sterile cellophane”.

L266. Change to “dia. 3 mm”.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

**********

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Submitted filename: Comments-PONE-D-22-11451.docx

Click here for additional data file.

9 Aug 2022

Response to Reviewer#1’s comments

We would like to thank Reviewer#1’s thorough work, important comments and suggestions. We think that the manuscript has significantly been improved with the implementation of the recommended changes done. All changes in the manuscript are highlighted in red. Please find below our responses to Reviewer#1’s comments (in bold).

Reviewer #1: Manuscript entitled "Mycoparasitic and growth inhibition capabilities of Clonostachys rosea isolates against grapevine trunk diseases fungal pathogens suggest potential for biocontrol" mainly talked about C. rosea isolates exhibited promising biocontrol ability against some grapevine trunk diseases. The manuscript is well written, and the results provide useful information for expanding the pathogen range by C. rosea biocontrol. Some points need to be addressed before it can be publication.

1. Line 63-69. Comprehensive review articles should be cited for introducing Clonostachys rosea. e.g. Sun et al. 2020 "Biology and application of Clonostachys rosea".

The characterisation of C. rosea in the introduction was extended in the revised manuscript with a special focus on the biotechnological application of the fungus. Several additional papers were also cited.

2. Line 75 and 232. Identification of fungal strains needs combined morphological characteristics and molecular sequencing. In line 232, the author declared that the identification of fungal strains would depend on morphological and molecular method. However, in Line 75, the results were mainly focus on ITS amplication. Then what about the morphological characteristics of these fungi? Especially the five C. rosea stains, are their morphological characteristics were same to known C. rosea strains?

Yes, the morphological characteristics of the C. rosea isolates were in accordance with its previous description. Related text was added to the manuscript.

3. Table 2. Why the significance analysis was only used for pathogens of Eutypa lata, Phaeoacremonium minimum and Phaeomoniella chlamydospora, but not for other pathogens in this Table?

We used significance analysis to find out if a C. rosea strain is a more potent antagonist of a given pathogen than another C. rosea isolate. All data were analysed in this context, but in case of pathogens other than E. lata, P. minimum and P. chlamydospora there were no significant growth inhibition, or the C. rosea isolates did not differ significantly in the RGI% value.

4. Line 98. Changed "antibiotic" to "antagonistic".

The word is changed according to the suggestion.

5. Line 105. Overgrow is a very important phenomenon for mycoparasitism. In this study, the author found the growth rate of Diaporthe strains were nearly not affect by C. rosea. Therefore, how about the overgrow behavior for C. rosea and Diaporthe strains? Just like in Fig.1 c and d, both two strains were overgrowth each other, or only C. rosea overgrowth pathogen strains? From Fig.1, it same like both the two strains are overgrowth each other.

Dose the single inoculation of C. rosea and pathogen strains were set as control in this study?

Only the C. rosea strains grow over the Diaporthe spp. and B. dothidea colonies, the opposite can not been observed. Yes, single inoculated controls were also prepared, but have not been presented in the manuscript as figure. We thought that photographs of the dual cultures are appropriate to demonstrate the phenomena we wanted to show, and photographs of single inoculations would not provide additional information.

6. Figure 1, why only (b) E. lata was investigated at 8 dpi, but other pathogens were at 15 dpi?

The two different phenomena we wanted to demonstrate (antibiosis and overgrowth) were expressed at the highest level at different times in case of different pathogens. E.g. P. chlamydospore is very slow growing, therefore it needs more time for its mycelia to grow into the “effective antibiotic range” of C. rosea colonies. In case of B. dothidea and Diaporthe spp. antibiosis have not been detected and for the demonstration of overgrowth, older cultures were more suitable.

7. Line 169-172. The sentence needs rewrote as "Our report is the first on the antagonistic activity of C. rosea against E. lata. Contrary to the findings of Silva-Valderrama..."

Confrontation test in plate could reflect the antagonistic activity of C. rosea against pathogens. However, antagonism had several mechanisms, and produce antibiotic is only one of the mechanisms. Therefore, from the confrontation test result, the author could provide conclusion of C. rosea had antagonistic activity against E. lata, but not antibiotic activity. Also the next sentence, C. rosea could produce many compounds with fungicidal activity, is not appropriate.

Thank you for the suggestions, the text was rewritten according to your instructions.

Response to Reviewer#2’s comments

We are thankful for the overall positive opinion about our paper and the careful reading of our manuscript. We do believe that the manuscript has significantly been improved during the revision process and fulfills the requirements of publication. All changes in the manuscript are highlighted in red. Reviewer#2’s comments are in bold.

Reviewer #2: This paper described a study of determining the effectiveness of Clonostachys rosea in controlling fungal pathogens causing grapevine trunk diseases (GTDs). The authors observed significant pathogen growth inhibition in both in vitro and in planta conditions. The reported findings contribute to the collective knowledge of biocontrol agents of C. rosea, especially with regard to in planta conditions. However, the following areas should be mentioned to improve the manuscript:

1. In abstract, experimental data should be provided to support the results and conclusion.

A more detailed description of experimental conditions and results were included in the revised manuscript.

2. The authors identified eight pathogenic species of grapevine trunk diseases basing on the previous researches, therefore, the related references should be supplemented in the Table. Meanwhile, C. rosea strains were also isolated from the plants. We cannot regard them as beneficial microorganisms before a primary test of pathogenicity to grapevine is performed, especially some C. rosea isolates were noticed to significantly promote the growth of some GTD pathogens in vitro test (Table 2).

Suggested references were added to the table. Your concerns on the harmless of C. rosea to grapevines are understandable. However, we can assume the suitability of the application of this fungus on V. vinifera because of the following reasons:

1.) The C. rosea isolates were obtained from healthy plants

2.) There were no symptoms observed in our study on plants growing in media amended with C. rosea.

3.) C. rosea is widely used on several crops without any undesired effects.

Point 1. and 2. were mentioned the revised manuscript.

3. The authors should provide the context when presented the results. It is hard to fully understand the terms used in the results section, however, a short description of the study method and arrangement will help the readers to understand the data presented. Also, the results of Table 2 and Table 3 need to be clearly described in the text.

A short description of the experimental setups were added to the results section in the revised manuscript, and table 2/3 are described with more details in the text.

4. The authors thought the in planta and in vitro experiments suggest "in practice, antifungal compounds produced by C. rosea may be more important for effective biocontrol purposes than mycoparasitism". It seems arbitrary, for the fungus was mainly found colonizing in soil and the base of the grapevine. In that case, induced resistance to the diseases is more involved, which has been verified in previous studies. It should be mentioned in discussion.

Thank you for the suggestion! The possibility of BCA induced resistance is mentioned in the discussion section with a reference.

5. Standard three-line tables should be used in the manuscript with the SI units in the tables. The notes should be put below the table, separating with the title.

Tables were reformatted according to your suggestions.

6. Supplement statistical analysis in the tables.

Where there were no significant differences, statistical groups were not labelled in the tables. We tried to reflect these cases in the text.

7. The figures should be improved. Fig. 1, which C. rosea isolate(s) was used? It should be marked. Fig. 2, clarify the arrows and change the bars to a line instead of an area. Fig.3, Rewrite the title to show the research content and put "x106" in the title of Y-axis. Different colors for the five isolates are not necessary.

The suggested changes were done on the figures.

8. Necrotic lesions were presented when inoculated with the pathogens (Fig.4), and the authors also mentioned that "while mock-inoculated plants did not show this symptom". Here, an uninfected cutting should be presented together with the infected cuttings in the figure.

The suggested photographs were added to the figure.

9. In discussion, the authors thought "only the C. rosea cells were stained by the MTT viability dye, suggests the necrotrophic nature of the parasitism", and "lack of specific structures". However, different infection structures have been observed in some mycoparasites, including C. rosea.

Thank you for the suggestion, you are absolutely right. While we in fact did not observe specific infection structures, this does not mean that their lack indicates necrotrophic parasitism. The related sentence was removed from the manuscript.

10. The authors should provide more information about the grapevine samples where the isolates were separated, for example, the geographical location, cropping years, infected or not, status of soil.

The suggested information was added to the “Materials and methods” section.

11. In the section of methods, the experimental procedure should be described with more details, therefore, the readers can clearly follow your steps.

Additional details were added where it was possible.

12. The term of "sporulation rate" is not correct.

“Sporulation rate” was changed to “conidia production”.

13. The full names of genus should be used when first appear, then only abbreviations are required if not confused.

Corrections were done according to the suggestion.

Furthermore, the authors should consider the following grammatical revisions:

Thank you for the careful reading of our manuscript! All the grammatical corrections suggested below were done and the title was changed.

L2-4. I would suggest to change the title to "Mycoparasitism capability and growth inhibition activity of Clonostachys rosea isolates against fungal pathogens of grapevine trunk diseases suggest potential for biocontrol".

L14. Write the exact number of the pathogens instead of "several".

L17-18. Change to "to the pathogens of Botryosphaeria dothidea and Diaporthe spp.".

L19. Change "on in vitro grown colonies" to "on PDA plates".

L21-22. "by inoculating... into the growth medium" is not consistent with the method.

L31-32. Can be revised to "chlorosis and necrosis occurrence on leaves, young shoots deformation, and necrotic spots appearance on the berries."

L33-34. There are two mis-spelt words "widespred" and "occour", and the sentence should be revised to "... the sudden death of the infected part or the whole plant may occur...".

L47. Change "pest management" to "disease management".

L81. In Table 1 use "strain number" instead of "strain ID".

L86. It should be "Growth inhibitions of C. rosea strains ...".

L90. Change "percental radial growth inhibitions" to "growth inhibition rates".

L115. Change to "Mycoparasitism of ..."

L141. Cancel "were".

L143. Change "developed by" to "caused by".

L151. Change to "the development of vascular necrosis caused by...".

L167. Change "This indicates..." to "This suggests...".

L169-170. Change to "Our study is the first report..." .

L191. Cancel the word "therefore".

L199. Change to "this fungus was also reported in the tissues of grapevine".

L207. Cancel the word "species".

L211. Change to "but susceptible to mycoparasitism".

L218: Cancel the comma after the word "suggest".

L251-252. Change "on cellophaned water agar (2 %m/v)" to "on 2% water agar covered with a piece of sterile cellophane".

L266. Change to "dia. 3 mm".

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

19 Aug 2022

Mycoparasitism capability and growth inhibition activity of Clonostachys rosea isolates against fungal pathogens of grapevine trunk diseases suggest potential for biocontrol

PONE-D-22-11451R1

Dear Dr. Váczy,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Estibaliz Sansinenea

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

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The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

**********

29 Aug 2022

PONE-D-22-11451R1

Mycoparasitism capability and growth inhibition activity of Clonostachys rosea isolates against fungal pathogens of grapevine trunk diseases suggest potential for biocontrol

Dear Dr. Váczy:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

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on behalf of

Dr. Estibaliz Sansinenea

Academic Editor

PLOS ONE