Next Article in Journal
Effects of Various Environmental Conditions on the Growth of Amaranthus patulus Bertol. and Changes of Herbicide Efficacy Caused by Increasing Temperatures
Next Article in Special Issue
Unraveling the Association between Metabolic Changes in Inter-Genus and Intra-Genus Bacteria to Mitigate Clubroot Disease of Chinese Cabbage
Previous Article in Journal
Environmental and Animal-Related Parameters and the Emissions of Ammonia and Methane from an Open-Sided Free-Stall Barn in Hot Mediterranean Climate: A Preliminary Study
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Agronomy
Volume 11
Issue 9
10.3390/agronomy11091771
agronomy-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

The Influence of Temperature on the Growth, Sporulation, Colonization, and Survival of Trichoderma spp. in Grapevine Pruning Wounds

by
Guzmán Carro-Huerga
1,
Sara Mayo-Prieto
1,
Álvaro Rodríguez-González
1,
Samuel Álvarez-García
1,
Santiago Gutiérrez
2 and
Pedro A. Casquero
1,*
1
Grupo Universitario de Investigación en Ingeniería y Agricultura Sostenible (GUIIAS), Instituto de Medio Ambiente, Recursos Naturales y Biodiversidad, Universidad de León, Avenida Portugal 41, 24071 León, Spain
2
Grupo Universitario de Investigación en Ingeniería y Agricultura Sostenible (GUIIAS), Área de Microbiología, Escuela de Ingeniería Agraria y Forestal, Campus de Ponferrada, Universidad de León, Avenida Astorga s/n, 24400 Ponferrada, Spain
*
Author to whom correspondence should be addressed.
Agronomy 2021, 11(9), 1771; https://doi.org/10.3390/agronomy11091771
Submission received: 4 August 2021 / Revised: 30 August 2021 / Accepted: 31 August 2021 / Published: 3 September 2021
(This article belongs to the Special Issue Microbial Control of Crop Diseases: Limitations and Optimizations)
Browse Figures
Versions Notes

Abstract

:
Trichoderma is a genus of fungi used for the biological control of plant diseases and a large number of its bio-formulates are available in the market. However, its efficacy under field conditions remains unclear, especially for the protection of grapevine plants against Grapevine Trunk Diseases (GTDs). These diseases are caused by a complex of fungal pathogens whose main point of entrance into the affected plants is through pruning wounds. In this research, different Trichoderma native strains have been evaluated according to their ability to grow at different temperatures and their capacity to colonize pruning wounds in adverse climatic conditions. Strains from section Trichoderma have adapted to cooler conditions. On the other hand, strains from clade Harzianum/Virens grow at higher temperatures. However, differences can also be found between strains inside the same clade/section. Native strains were able to colonize more than 70% of vine pruning wounds in winter conditions. The Trichoderma strain T154 showed a significantly higher re-isolation degree from vine plants and its concentration was optimized for spraying onto vine plants. In conclusion, Trichoderma native strains are better adapted to survive in a changing environment, and they could give better protection to grapevine plants in co-evolution with each specific vineyard.

1. Introduction

Trichoderma spp. Pers. are ubiquitous filamentous fungi, some of which are able to produce beneficial outcomes in crop production and have been used as natural pathogen antagonists in agricultural fields for a long time [1]. Some of them have been isolated and selected as biological control agents to be used in integrated pest management strategies to reduce the use of chemical pesticides [2]. This selection is due to their capacity to use diverse mechanisms involving the microorganisms themselves, their genes, and their metabolites [3].
Therefore, introducing Trichoderma as a biological agent in viticulture could have a positive impact on grapevine plants [4]. Trichoderma has been proven to be an effective biological control agent (BCA) against Botrytis cinerea Pers. together with integrated pest management strategies [5]. This biological control agent also reduced Grapevine Leaf Stripe Disease (GLSD) symptoms in vine plants over a long period of time [6]; these symptoms are associated with fungal species of Phaeoacremonium minimum, and other species of Phaeoacremonium and Phaeomoniella chlamydospora [7]. Moreover, this fungus can induce resistance against downy mildew caused by Plasmopara viticola (Berkeley and Curtis) and Berlese and de Toni in grapevine plants [8]. Moreover, its secondary metabolites are able to control some other pathogens such as Neofusicoccum parvum (Pennycook and Samuels), P.W. Crous, Slippers and A.J.L. Phillips and Eutypa lata (Pennycook and Samuels) Crous, Slippers, and A.J.L. Phillips [9].
In fact, in the last few years, species of Trichoderma have been widely studied for their action against Grapevine Trunk Diseases (GTDs), which has enabled important advances in terms of reducing the impact of GTDs [4]. This complex of diseases can be divided individually into six different types of disease: Petri disease, Black foot, Botryosphaeria dieback, Eutypa dieback, Phomopsis dieback, Esca, and Grapevine Leaf Stripe Diseases, and each of them are associated with a complex of fungal pathogens that cause these diseases [10]. Petri disease is caused by Phaeomoniella chlamydospora, 29 species of Phaeoacremonium, Pleurostoma richardsiae, and 6 species of Cadophora [11,12,13]. Black foot is caused by up to 24 species belonging to the genera Campylocarpon, Cylindrocladiella, Dactylonectria, Ilyonectria, Neonectria, and Thelonectria [14,15,16]. Esca and Grapevine Leaf Stripe Disease are described as successive invasions of fungi, where the pioneer fungi are P. chlamydospora and/or species of Phaeoacremonium and after that, basidiomycetous species continue to colonize the grapevine wood [10]. Phomopsis is caused by the genera Diaporthe but seven species have been described as pathogenic on grapevine wood [17,18]. Botryosphaeria dieback is mainly associated with taxa from the genera Botryosphaeria, Diplodia, Dothiorella, Lasiodiplodia, and Neofusicoccum in grapevines [19,20,21]. Eutypa dieback is caused by species from the genera Diatrypaceae and the most virulent and common is Eutypa lata [22,23]. All spores of these GTD fungi can infect grapevine plants and annual pruning wounds are the primary point of entry [10].
However, Trichoderma species do not always act as effective biocontrols for grapevine diseases and fungal species of GTDs. For example, Trichoderma did not significantly reduce the incidence of black foot (the majority of fungal species associated to D. torresenis, D. macrodidyma, Dactylonectria novozelandica, and Ilyonectria liriodendri) in comparison to other treatments [24]. In addition, no positive effects were obtained in relation to the shoot and root weight in grapevine plants including a non-significant reduction in symptoms caused by black foot disease (caused by Dactylonectria torresensis, Dactylonectria macrodidyma, Ilyonectria liriodendri, and Dactylonectria alcacerensis) and Petri disease (caused by Cadophora luteo-olivacea, Phaeoacremonium minimum, and Phaeomoniella chlamydospora) and diseases in all BCAs assayed such as Trichoderma, Streptomyces, Pythium, Pseudomonas, and Bacillus spp. [25]. Different Trichoderma bio-formulated-based treatments applied on grapevine roots reduced their weight significantly during the first year, and these fungi were not able to prevent infections caused by the black foot disease pathogens identified as Campylocarpon spp., Dactylionectria, Ilyonectria spp., and Ilyonectria liriodendri [26]. Moreover, some experiments that compared Trichoderma bio-based commercial agents and chemical products have concluded that chemicals are more efficient in terms of pathogen control (Eutypa lata, Diplodia seriata, and Phaeomoniella chlamydospora) than the Trichoderma biological control agents assayed [27,28].
The different effects observed could be strongly influenced by the disease tetrahedron concept, where the pathogen, host, environment, and biotic factors play a key role [29]. Among them, environmental factors are thought to have a major impact on disease development [30]. The effects of temperature are considered to be an important parameter for proper biocontrol activity [31]. Recent research has found that temperature and moisture conditions affect the growth and survival of the theoretical BCA significantly. They could be considered key factors for efficient biocontrol [32]. Moreover, another experiment showed that temperature and moisture levels could be used to make good predictions for effective biocontrol using Trichoderma among other BCAs against Botrytis cinerea in grape berries [33]. Moreover, an increase of 5 °C can significantly influence the response of Trichoderma in relation to the induction of the plant response in tomato plants [34]. All these environmental factors and the alteration in temperatures due to climate change [35] represent major challenges in wine production. Temperatures are increasing worldwide, and this change is leading to a lack of water, shifts in the ripening phase, and modifications in grape composition, along with other changes such as the increase in extreme climatic events [36]. Rising temperatures lead to an advanced phenology and sugar accumulation in Vitis vinifera L. [37]. On the other hand, it has been reported that the negative effects caused by increasing temperatures and higher CO2 could be mitigated by a higher ultraviolet-B radiation that could modulate the accumulation of sugars and upregulate anthocyanin biosynthesis in V. vinifera cv. Tempranillo [38]. Furthermore, many other harmful effects have been described and have pointed to the need to use new suitable molecular techniques in order to mitigate these effects [39].
Currently, many studies are being conducted to characterize the effects of abiotic factors due to climate change which involve changes in the physiology, phenology, and harvesting of grapevine plants [40,41,42]. Some studies concluded by emphasizing the importance of evaluating the effects of these changing environmental conditions on the biological control microorganisms in grapevine plants, in order to predict their possible adaptations to the changing temperatures and relative humidity conditions [43,44,45]. Most Trichoderma species are mesophilic, and low winter temperatures might be a problem for their biological activity and their emergence, which is also due to dry conditions [31]. To summarize, it is important to evaluate the influence of different temperature conditions of Trichoderma on the different species, and isolate selected ones as putative biocontrol agents. These assays must be carried out previously under semi-field conditions before being sprayed in the field, because an effective biocontrol activity in in vitro conditions could lead to failure in the field due to a deficient adaptation to the environment, and as a result prevent the growth and the performance of any biological activity. The importance of using native strains to obtain a better protection for grapevine pruning wounds could be a key factor due to the possibility that they possess better adaptative responses to these conditions.
Based on this, we described and assessed different studies that would be required in order to carry out different assays to characterize a biological control agent under the current changing environmental conditions before being sprayed in the field. These experiments have been divided into two stages: (i) testing different Trichoderma strains at different temperatures and their effects on fungal mycelia and spore development to select the best strains, and (ii) evaluating the colonization of pruning wounds under winter conditions in the event of climate change.

2. Materials and Methods

2.1. Fungal Isolates

Four Trichoderma strains (T065, T071, T154, and T214) were obtained from the culture collection of the Plant and Pest Diagnostic Laboratory under the accession numbers ULET065, ULET071, ULET154, and ULET214, respectively (University of León, León, Spain). All isolates were selected due to their biocontrol activity and were isolated from vineyards from Castilla y León (Spain) (Table 1). Trichoderma spp. T065 and T214 were selected based on their high potential to produce secondary metabolites; T214 showed a remarkable mycoparasitic activity against the pathogenic fungus Phaeoacremonium minimum (unpublished data); Trichoderma T071 is effective against the Xylotrechus arvicola insect pest [46]; and Trichoderma sp. T154 also possesses significant mycoparasitic activity against P. minimum [47].

2.2. Fungal Identification

The identification of Trichoderma was performed using sequences of five housekeeping genes (act1 (encoding for the actin); cal1 (calmodulin); fas1 (fatty acid synthase alpha subunit); rpb2 (RNA polymerase 2nd largest subunit), lcb2 (sphinganine palmitoyl transferase subunit 2); and tef1 (translation elongation factor 1-alpha)) retrieved from the genome sequence of T065 and T214 isolates. BLASTn software was used to compare these six genes of the genome of T. harzianum CBS 226.59 against the genome sequences of T065 and T214, using the sequences of fungal homologous genes as queries (Supplementary Material Appendix). Finally, intron regions from the six housekeeping genes obtained were manually removed, the resulting coding sequences were compared to NCBI Genbank (National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov, accessed on 21 July 2021) databases using the BLAST program (http://www.ncbi.nlm.nih.gov/BLAST, accessed on 21 July 2021), and a phylogenetic tree was performed as indicated in the legend of Supplementary Material Figure S1. In conclusion, Trichoderma T065 belongs to Section Trichoderma (ST) and Trichoderma T214 belongs to Clade Harzianum/Virens (HV) (Supplementary Material Figure S1).
Trichoderma T154 was classified into Clade Harzianum/Virens (HV) [47] and Trichoderma T071 was assigned to Section Trichoderma (ST) [46]. The criteria followed for this assessment was applied according to Kubicek et al., 2019 [48].

2.3. Growth Evaluation

An in vitro evaluation of the four potential BCAs (T065, T071, T154, and T214) was carried out to compare the effects of temperature in cultures grown on 90 mm Petri dishes containing a potato dextrose agar (PDA) medium (Sigma-Aldrich Chemie GmbH, Steinheim, Germany). Mycelial development and sporulation were evaluated in order to determine their capacity to face constantly changing environmental conditions. This protocol was designed to evaluate the colonization capacities of the Trichoderma native strains with regard to biocontrol in relation to GTDs, protecting vine pruning wounds in our local region (Castilla y León, Spain), and trying to avoid the entrance of GTD pathogens, the most harmful diseases in viticulture and the ones without any available effective control [10]. First, the most common fungi associated with the species of GTDs in Castilla y León region (Spain) were selected and identified according to Cobos et al., 2007 [49]. The range of growth of these pathogens was then retrieved from the bibliography (Table 2) and a range of temperatures from 4 °C to 35 °C was chosen to test the ability of Trichoderma native strains to grow at these temperatures.
Mycelial growth was assessed as follows: each plate was inoculated with one agar plug (7 mm diameter) of a Trichoderma strain collected from a 7-day-old culture grown on a PDA. Plates were incubated in the dark at 4, 15, 25, and 35 °C. Mycelial growth was assessed daily except for the plate at 4 °C that was evaluated after six weeks. The experiment was performed with a completely randomized design where each plate was considered a biological replicate, and a total of three replicates were evaluated; moreover, this experiment was conducted twice in order to ensure reproducibility. Data from both experiments were combined (p < 0.05), and a total of six biological replicates were used for the statistical analysis.
Spore production was evaluated for each Trichoderma strain from a 7-day-old culture grown on a PDA medium grown at 25 °C. Spore suspensions were prepared from these cultures by dislodging the spores with a sterile loop and filtering them through a sterile cheese cloth to remove mycelium fragments. The spores were counted using a haemocytometer. Two technical replicates per plate and three replicates were evaluated. This experiment was performed twice.
In addition, the evaluation of the mycelial growth from the spores was also assayed at low temperatures. They were incubated at 4 °C and 15 °C, and two concentrations of spores were evaluated, 2 × 10−6 and 2 × 10−7 colony forming units (CFU)/mL. Mycelial growth was assessed daily at for the plate at 15 °C and for the plate at 4 °C it was evaluated after six weeks. In all cases, radial growth was evaluated usually after seven days, which is the time that the fastest fungus needed to completely colonize a Petri dish.

2.4. Evaluation of Trichoderma Survival over Pruning Wounds in Planta

The survival of the Trichoderma strains was assessed over pruning wounds in planta under semi-field conditions. This condition involved potted vine plants in a protected environment. Trichoderma strains T071 and T154 were selected for this experiment; in the case of T071, this strain was selected due to its capacity to grow at low temperatures, and T154 was selected for its ability to produce a high number of spores and also for its ability to grow significantly at higher temperatures. They were grown on a PDA medium at 25 °C in the dark for 7 days. It was decided to inoculate a total amount of 50 µL in a concentration of 2 × 10−6 CFU/mL and 2 × 10−7 CFU/mL suspensions of T071 and T154 which were sprayed over a pruning wound to prevent the entrance of fungal pathogens within a period of 24 h after being pruned [53].
One-year-old dormant grapevine grafted plants of the Tempranillo/110 Richter combination from Vivai Cooperativi Rauscedo (Rauscedo, Italy) were potted in June 2017. The pots contained a sterile mixture of vermiculite and peat (1:1 v/v). Each plant was considered a biological replicate and eight biological replicates per treatment were performed. These plants grew during the vegetative season and, after that, during dormancy, when the plants were pruned to one spur and Trichoderma was later sprayed in November 2017. The experiment was carried out in controlled conditions where warmer and dryer conditions were used in order to simulate climatic change parameters. A thermohygrometer (Electroson, Valladolid, Spain) was used (defined as a local weather station) to measure these conditions and was compared to the standard automatic weather station located close to the trial sites. Data were retrieved from the website (http://www.inforiego.org/opencms/opencms, accessed on 21 July 2021).
After 12 weeks of inoculation (February 2018), the effectiveness of the colonization was evaluated following this protocol: eight wood chips were cut (approx. diam. 1–2 mm; approx. length 0.5–1 cm) over the inoculation point, and placed 3, 6, 9, 12, and 15 cm from the inoculation point, over Rose Bengal agar (RBGA, Conda Laboratory, Torrejón de Ardoz, Madrid, Spain) plates. They were incubated at 25 °C in darkness for seven days and the growing isolates were morphologically identified [54]. The colonization index was determined as the mean percentage of wood chips (8 chips × 8 plants = 64) placed by the inoculated fungus in order to avoid fungal pathogens that could develop GTDs.

2.5. Statistical Data Analysis

To compare the degree of colonization between the isolates, an evaluation of growth, spore production, and pruning wound colonization were performed using a simple analysis of variance (ANOVA). The Duncan post hoc test at p = 0.05 was evaluated. Analyses were conducted using Statistics for Windows Version 26.0, IBM Corp., Armonk, NY, USA.

3. Results

3.1. Evaluation of Fungal Growth

Mycelial growth was evaluated at temperatures ranging from 4 °C to 35 °C and different behaviours were identified depending on the Trichoderma strain. At 4 °C, the total radial growth over the PDA plate after 6 weeks of Trichoderma T071 was significantly greater (p < 0.05) than the other Trichoderma strains (Figure 1). T154 did not grow at all at this temperature, and no significant differences were seen compared to the Trichoderma T065 strain. However, the rest of the Trichoderma strains exhibited very small growth (Figure 1).
Afterwards, they were grown at 25 °C and were all able to colonize the plate after 7 days (data not shown).
At 15 °C, strains T065, T071, and T214 showed a significantly higher growth (p < 0.05) in comparison to T154 on day six (Figure 1). The lag phase (little or no observable growth) was considered as 1 day. At 25 °C, no significant differences were found between them (Figure 1). On day three, all the Trichoderma strains were able to entirely colonize the plate. At 35 °C, the growth of T154 was significantly greater (p < 0.05) than the rest of the Trichoderma strains (Figure 1). T214 showed a moderate growth rate and a significantly greater rate of growth than T065 and T071. These last two strains were not able to grow at 35 °C, thus showing a lag phase for the whole experiment (7 days). (Figure 2).
Spore production was assessed at 25 °C in darkness in order to determine their suitability for scale production. The Trichoderma strain T154 produced a significantly higher (p < 0.05) number of spores in comparison to the rest of Trichoderma strains (Figure 3). T214 also produced a significantly higher production of spores than T065 and T071. The lowest value of spore production was found for T065 and T071 and no significant differences were found between them.
The mycelial growth of the Trichoderma spp. in the PDA plates was evaluated from 5 microliters from two different spore concentrations (2 × 10−7 CFU/mL and 2 × 10−6 CFU/mL) and in combination with two low values of temperatures (15 °C and 4 °C). At 15°C and 2 × 10−7 CFU/mL, the Trichoderma strains T065 and T071 showed a growth rate significantly higher (p ≤ 0.05) than the others. When the concentration was 2 × 10−6 CFU/mL and the temperature was 15 °C, the Trichoderma strain T071 had a significantly greater growth rate (p < 0.05) in comparison to the other Trichoderma strains. The lag phase was 2 days for all of the strains. (Figure 4). At 4 °C and using both concentrations, none of the strains were able to develop a mycelium or had observable growth after 6 weeks.

3.2. Evaluation of Survival over Pruning Wounds in Planta

The Trichoderma strains T071 and T154 were inoculated over pruning wounds to colonize and prevent the entrance of other fungal pathogens (Figure 5A,B) at 6 °C and a relative humidity of 56.00%. Both strains were able to survive after 12 weeks in in planta conditions during winter, from November 2017 to February 2018. Data from the minimum mean temperatures were selected as references to evaluate possible climate change events. Weather conditions recorded in a local weather station had a higher temperature media of 4.06 °C compared to a standard weather station and a lower than standard relative humidity media of 27.30%. The average temperature and relative humidity per week are recorded in (Figure 6A,B).
The evaluation of the pruning wounds to study the capacity of Trichoderma to colonize the pruning wounds was performed by re-isolating the fungal strains from the inoculation points 12 weeks after the inoculation at a concentration of 2 × 10−6 CFU/mL from both the T154 and T071 strains.
The percentage of the re-isolation of T154 was 89.58%, a value significantly higher (p < 0.05) than that observed for T071, which reached 70.31% (Figure 7). Thus, T154 was chosen as more suitable strain for pruning wound colonization in order to avoid the entrance of possible pathogens that could lead to GTDs. Furthermore, a colonization of 91.67% was found for T154 when a concentration of 2 × 10−7 spores/mL was used. However, no significant differences were found when compared with the concentration of 2 × 10−6 spores/mL in terms of re-isolation (Figure 7).
Trichoderma strains were recovered 3 cm above the inoculation point in one of the eight vines for both of the strains. However, below the inoculation points at 6, 9, 12, and 15 cm, it was not possible to re-isolate Trichoderma.

4. Discussion

This work was conceived to try to efficiently evaluate the persistent performance of biological agents in semi-field conditions in winter to colonize pruning wounds as an assessment of a niche exclusion mechanism in order to block the entrance to other hypothetical fungal pathogens trough the xylem and phloem vessels. This is caused due to most biological control agents after obtaining positive laboratory tests. A poor efficacy rate was found for many biological control agents during the field tests; this has been attributed to host and abiotic factors that favour pathogen development [53,55]. In this article we evaluated abiotic factors and V. vinifera cultivar adequation before moving on to field conditions.
Under in vitro conditions and over grapevine plants, all Trichoderma strains showed different adaptations to different temperature conditions, demonstrating similar patterns according to its clade or section that it belonged to genetically. From the four Trichoderma strains evaluated under in vitro conditions, two of them (T154 and T071) were chosen to perform semi-field condition assays. They showed a good rate of survival over the inoculation points during the winter season over canes of grapevines, but T154 was re-isolated at a significantly higher rate than T071.
Firstly, in vitro evaluations of T065, T071, T154, and T214 led to the observation that they behave as mesophilic fungi because they are able to grow at 25 °C, which is an optimal temperature, as most of Trichoderma spp. do, as previously described [56,57]. T154 and T214 that belong to clade HV showed a good response at 35 °C in comparison to the T065 and T071 strains that are classified in the section SL, which have been described as able to grow under low temperatures [58,59]. Trichoderma harzianum have been described as a complex species that are adapted to warm climates [60]. At 15 °C, T154 showed a significant lower growth in comparison to the rest of fungi, confirming this trend. At 4 °C, T071 showed a significant growth but at a much lower level than other cold tolerant Trichoderma strains [61]. This Trichoderma strain (T071) was the only one to have a significant growth rate in comparison to the others, which were able to grow slowly at a very low temperatures among all the strains analyzed in this work.
Moreover, the rate of spore production is an important characteristic since most commercial bio-formulated Trichoderma are presented in spore suspensions [2]. T154 and T214 that belong to the HV clade had a significantly higher production of spores than those belonging to section SL. In order to scale and optimize mass production, different substrates should be tested, such as agro-wastes, in order to reduce production costs, and also to obtain a higher and easier obtention of CFUs [62].
The performance of the spores was also evaluated at low temperatures in order to simulate in vitro the capacity to colonize a pruning wound at a low temperature during the winter season, i.e., at 15 °C. The same trend was observed with Trichoderma strains that belonged to section SL that had a significantly higher growth rate in PDA plates in comparison to species of clade HV. Thus, the use of biological control strains that belong to section SL could reduce the problems associated with low temperatures during winter, as result of the lower activity of the BCA [31]. Previous reports have also described a higher proportion of cold-tolerant strains among those belonging to the T. viride strains in comparison to the T. harzianum strains, and a higher optimal growth temperature for the species that belongs to clade HV [63,64]. All Trichoderma strains were able to sporulate and grow at 15 °C. Thus, all of them could eventually colonize and grow in winter conditions.
In conclusion, the in vitro assays, the range of temperatures assayed with these two different section/clades, and our results suggest that using the Trichoderma species belonging to section SL for the winter season and clade HV for the summer could be an option in order to optimize their capacities. Moreover, a combination of both kinds of Trichoderma strains could diminish the effects of climate change in viticulture [65].
The taxonomy of these strains is also an important issue. Thus, T065 and T071 have been identified as T. gamsii. This species was reassessed in 2006 [66] as a new species that belongs to section SL. Other T. gamsii strains have been described as effective BCAs in tomatoes [59,67]. Moreover, they have been described as effective BCAs against grapevine trunk diseases such as Esca—specifically for reducing the external symptoms of grapevine leaf stripe diseases, combined with T. asperellum and T. gamsii [6]. T. harzianum is a complex species distributed worldwide and is a very common BCA. However, not all T. harzianum strains can be described as efficient BCAs. Previously, T154 has been tested against P. minimum as an effective BCA that uses niche exclusion mechanisms for biocontrol [47], and T214 was shown to have a significant antifungal activity in our case (data not shown). These two strains belong to the clade HV that possesses a complex taxonomy and has not been fully identified due to its coexistence and interaction with all these organisms that together create this clade and have a lack of strict genetic borders between them [68].
The colonisation of grapevine pruning wounds is a key factor to ensure the effectiveness of these strains as BCAs against pathogens that could develop GTDs or a fungal infection after evaluating temperature performance. Annual pruning wounds are the main point of entry for pathogens that cause GTD pathogens [10]. Thus, a good colonization of Trichoderma strains would prevent the penetration of the main pathogens such as Diplodia seriata, Phaeoacremonium minimum, or Phaeomoniella chlamydospore, among others. An assay that evaluates the persistence of Trichoderma strains over a pruning wound will help to decide if these strains are suitable to be sprayed in field conditions. In order to reduce natural infection, early pruning (November) is recommended in combination with a biological or chemical wound protectant to reduce the risk of infection of Eutypa lata, Neofusicoccum parvum, Botryosphaeria dothidea, Phomopsis spp., Cryptovalsa ampelina, Phaeomoniella chlamydospore, Diplodia seriata [69], and the genera Botryosphaeria, Cadophora, Cryptovalsa, Cytospora, Diaporthe, Diplodia, Eutypa, Neofusicoccum, Phaeoacremonium, and Phaeomoniella [70] in comparison to late pruning (February) [57,58]. Moreover, delayed pruning may result in the sap-flow washing away any pruning wound protectant [53]. Thus, early pruning and immediate Trichoderma application is the most advisable practice.
In this semi-field experiment, the incidence of Trichoderma was evaluated after 12 weeks of inoculation. Its persistence over grapevine wounds was significantly higher at 89.58% when sprayed with T154 at 2 × 10−6 CFU/mL, in comparison to 70.31% for T071 at the same concentration of spores. Niche exclusion was described as one of the mechanisms used by T154 [47] in order to biocontrol P. minimum and it could be a reason for its successful colonization. Moreover, its capacity to produce a greater number of spores and its mycoparasitic activity would point to this strain as a suitable BCA [71]. In the present work, Trichoderma spores were sprayed during the dormant vine period and 24 h after pruning as is recommended [53]. No sap bleeding was detected after pruning and no rain fell after application. Similar results were obtained for the T. atroviride isolate UST1 (highest rate of incidence corresponded to 90.00%) and Eco-77 based on T. harzianum (highest rate of incidence corresponded to 87.00%), and no differences were found between the strains of clade HV and section SL [53].
Previous studies have determined that the percentage of prevalence recovery after eight months can vary according to the Trichoderma strain and the grapevine cultivar from 82.5% to 0.00% of mean prevalence [72]. Moreover, the highest incidence recovery of Trichoderma in a pruning wound was 59.03% of the colonization of the pruning wound after seven months [73]. Another experiment obtained a 33.41% incidence recovery in pruning wounds as the highest value after 8 months [74]. Trichoderma bio-based commercial products authorized currently in Spain such as Vintec® (T. atroviride SC1) and Esquive were assayed and the percentage of recovery after 12 months was as high as 13.5% and 9.8%, respectively, from pruning wounds [28]. In our case, the incidence of the recovery of Trichoderma was obtained after 3 months in semi-field conditions. Thus, further investigations would be needed to analyze a wider range of months (7–8) in a Vitis vinifera cv. Tempranillo field plant, in order to confirm the data from the in vitro and semi-field assays. This experiment revealed that Trichoderma strains were not able to penetrate but were able to colonize the pruning wound at a high percentage rate during the period of maximum risk of infection [75]. The period of evaluation in planta needs to be longer in order to validate the movement through the plant, as was found in the Trichoderma strain AG1 after 20 months of inoculation, where it was found 6 cm far from the inoculation point [76].
Several factors have influenced these results. One of them is nutrient availability. Grapevine canes possess cellulose, hemicellulose, and lignin [77] in comparison to alpha polysaccharides that contain PDAs such as dextrose and potato extract (PDA, Sigma-Aldrich Chemie GmbH, Steinheim, Germany). This suboptimal nutrient availability and the effect of temperature can lead to a less effective substrate colonization [78] and it could be the reason for the lack of penetration through the grapevine cane. Temperature and moisture also play an important role during the colonisation along the cane, and higher temperatures and a humidity relative lower than 90% could generate lower rates of colonization for the Trichoderma species [33]. During the experiment in semi-field conditions, the media of the minimum temperatures barely reached 4 °C and the relative humidity minimum from week 2 to week 12 was lower than 50%, which led to very adverse conditions that did not allow it to grow far from the inoculation point. This follows the studies of John et al., 2008 [76], which obtained a colonization of 3 cm after 4 months. Moreover, T071 showed a significantly lower colonization over the grapevine cane despite having a better growth in in vitro assays under low temperatures. A different degree of colonization of the Trichoderma species was achieved in different grapevine varieties [72]. Thus, it is possible that T071, isolated from soil, would have a lower capacity of colonization than T154, isolated from the grapevine bark of V. vinifera cv. Tempranillo. These results are in agreement with data from Mayo-Prieto et al., 2020 [79], where Trichoderma native strains isolated from soil produced a higher percentage of germination and better agronomic parameters in comparison to those that were isolated from seeds. Both results demonstrate the importance of the source used for the fungal strain isolation. However, further research under field conditions is needed in order to confirm an early pruning time as a good option in order to combine it with the shorter period of infection of GTDs [70].
In addition, other important issues to take into account are the environmental conditions at the time of application. The temperature recorded was 6 °C with 56% relative humidity. Most experiments have not published these data [72,73,76,80] except Martínez-Díaz et al., 2020 [28], where the daily mean temperature was 6.5 °C and 8.2 °C during the week of the trials and Trichoderma did not colonize well. This is in contrast to the study by Mutawila et al., 2015 [53], where an average temperature of 15.6 °C and 12.9 °C was achieved during the first year at inoculation time and 12.0 °C and 13.4 °C during the second year. According to the relative humidity, the values of 59% and 65% during the first year of the experiment were obtained and 69% and 74% were recorded during the second season. In this case, Trichoderma obtained a high rate of re-isolation from the pruning wounds. Probably, higher temperature values and the relative humidity could influence the superior performance of Trichoderma colonization. Rainfall events were also recorded, and this factor is important for field experiments after pruning as the probability of infection increases due to GTDs [69,70]. Furthermore, it strengthens the idea that it is necessary to: (I) study the capacity of growing in these environmental and in vitro conditions and the same available nutrients; (II) measure environmental variables with the aim of characterizing the optimal conditions [81] for a good colonization, combining the knowledge of microbiology and engineering.
Regarding the concentration of spores, a greater range of spores at the inoculation point can vary in the vine plants from different Trichoderma species [27,72,74,76,80,82,83]. T. harzianum AG1 had the highest value, with a concentration of 10−9 spores/mL [80] and T. harzianum Biotricho had the lowest value, where 4.8 × 10−4 spores/mL were inoculated [74]. According to this range, it was decided to test 2 × 10−6 CFU/mL and 2 × 10−7 CFU/mL as two possible concentrations. Excessive concentrations of Trichoderma could have negative consequences for yield production [84]. Thus, the optimization of these parameters could help obtain a more sustainable biological product. In this case, no significant differences were found under in vitro or semi-field conditions in Trichoderma sp. T154 when these two concentrations were evaluated. The volume of the spore suspension could be closely linked to this issue. In our case we inoculated 50 µL in each pruning wound in 0.5–1 cm of the cane diameter in order to optimize the inoculation as much as possible. The inoculation of Trichoderma involves spraying a suspension of 2 mL [74] and other liquid formulations were applied using a 500 mL hand sprayer [28,53], and the results were totally different.
Another issue of discussion is the effectiveness of native strains versus commercial ones. In this regard, some previous reports have not found significant differences between them in terms of biological control efficacy [85]. Despite this, the studies performed with Trichoderma native strains isolated from bean soils showed an excellent in vitro biocontrol against Rhizoctonia solani J.G. Kühn [86]. Furthermore, native Trichoderma were able to colonize pruning wounds against P. minimum [47]. However, on the contrary, studies carried out with some commercial Trichoderma-based bioproducts concluded that they did not show a good plant colonization ability under the selected conditions [28]. Similar controversial results have been reported for other non-fungal biological control agents, as in the case of Streptomyces [25,87]. These data show how important it is to continue studying the biotic and abiotic factors that affect the success of a good colonization as a key step to a good biocontrol activity.
The studies carried out in the present work, including the determination of the range of temperatures under which the different Trichoderma strains can grow, and also the confirmation of the establishment of these strains over pruning canes, will allow a reduction in the negative impacts of pathogens in different currently changing abiotic conditions and would ensure the one health concept by which humans, animals, and plants interact based on the use of effective biological control products. Thus, the main result of this article is the description of a method for evaluating the Trichoderma response to different environmental conditions, which would allow us to determine the suitability of the analyzed strains to be used in a background of climate change. As a concluding remark, our results indicated that the Trichoderma native strains show a good ability of colonization and a good response at different combinations of temperature. They could give better protection to grapevine plants in co-evolution with each specific environment.

5. Conclusions

Although Trichoderma strains from section Trichoderma were better adapted to cooler conditions and the others from clade Harzianum/Virens showed a greater capacity for growth under warmer temperatures during in vitro tests, Trichoderma T071 selected from the section Trichoderma had a significantly lower capacity to colonize the pruning wounds than Trichoderma T154 that belongs to Clade Harzianum/Virens during winter conditions. None of them were able to penetrate the grapevine cane but they were able to colonize successfully pruning wounds under adverse climate situations. Trichoderma T154 had a significantly higher colonization rate, probably due to its origin from grapevine wood and its effective mechanism of niche exclusion. Further studies are needed to evaluate these strains for longer periods of time in order to contrast temperature adaptation and test them against the main pathogens that cause grapevine trunk diseases infections.

Supplementary Materials

The following are available online at https://www.mdpi.com/article/10.3390/agronomy11091771/s1, Figure S1: Trichoderma species phylogenetic tree. Supplementary Data Appendix: Sequences retrieved from the genomes analyzed in this work.

Author Contributions

All authors contributed to the study conception and design. Material preparation, sampling and data collection were performed by G.C.-H., S.M.-P. and Á.R.-G. Statistical analysis was carried out by G.C.-H., S.Á.-G. and P.A.C. G.C.-H., S.Á.-G., Á.R.-G., S.M.-P., S.G. and P.A.C. performed the data interpretation and manuscript preparation. Supervision of all study was performed by S.G. and P.A.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by project GLOBALVITI ‘Solución global para mejorar la This research was funded by the Centro para el Desarrollo Tecnológico Industrial–CDTI—(Madrid, Spain) for the project GLOBALVITI project (CIEN Program) IDI-20160746, and the project LowpHwine (CIEN Program) IDI ‘Estudio de nuevos factores relacionados con el suelo, la planta y la microbiota enológica que influyen en el equilibrio de la acidez de los vinos y en su garantía de calidad y estabilidad en climas cálidos’—and the grant awarded to GC-H comes from the Ministry of Education, Culture, and Sport (SPAIN), grant number (FPU15/04681) and Bodega Pago de Carraovejas S.L.U.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

We thank technical assistance of Javier Saiz Gadea.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

References

  1. Lorito, M.; Woo, S.L.; Harman, G.E.; Monte, E. Translational research on Trichoderma: From ’omics to the field. Annu. Rev. Phytopathol. 2010, 48, 395–417. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  2. Woo, S.L.; Ruocco, M.; Vinale, F.; Nigro, M.; Marra, R.; Lombardi, N.; Pascale, A.; Lanzuise, S.; Manganiello, G.; Lorito, M. Trichoderma-based products and their widespread use in agriculture. Open Mycol. J. 2014, 8, 71–126. [Google Scholar] [CrossRef] [Green Version]
  3. Vinale, F.; Sivasithamparam, K.; Ghisalberti, E.L.; Marra, R.; Woo, S.L.; Lorito, M. Trichoderma-plant-pathogen interactions. Soil Biol. Biochem. 2008, 40, 1–10. [Google Scholar] [CrossRef]
  4. Mondello, V.; Songy, A.; Battiston, E.; Pinto, C.; Coppin, C.; Trotel-Aziz, P.; Clément, C.; Mugnai, L.; Fontaine, F. Grapevine trunk diseases: A review of fifteen years of trials for their control with chemicals and biocontrol agents. Plant Dis. 2018, 102, 1189–1217. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  5. Pertot, I.; Giovannini, O.; Benanchi, M.; Caffi, T.; Rossi, V.; Mugnai, L. Combining biocontrol agents with different mechanisms of action in a strategy to control Botrytis cinerea on grapevine. Crop Prot. 2017, 97, 85–93. [Google Scholar] [CrossRef]
  6. Bigot, G.; Sivilotti, P.; Stecchina, M.; Lujan, C.; Freccero, A.; Mosetti, D. Long-term effects of Trichoderma asperellum and Trichoderma gamsii on the prevention of esca in different vineyards of Northeastern Italy. Crop Prot. 2020, 137, 105264. [Google Scholar] [CrossRef]
  7. Surico, G. Towards a redefinition of the diseases within the esca complex of grapevine. Phytopathol. Mediterr. 2009, 48, 5–10. [Google Scholar] [CrossRef]
  8. Perazzolli, M.; Roatti, B.; Bozza, E.; Pertot, I. Trichoderma harzianum T39 induces resistance against downy mildew by priming for defense without costs for grapevine. Biol. Control 2011, 58, 74–82. [Google Scholar] [CrossRef]
  9. Mutawila, C.; Vinale, F.; Halleen, F.; Lorito, M.; Mostert, L. Isolation, production and in vitro effects of the major secondary metabolite produced by Trichoderma species used for the control of grapevine trunk diseases. Plant Pathol. 2015, 65, 104–113. [Google Scholar] [CrossRef] [Green Version]
  10. Gramaje, D.; Urbez-Torres, J.R.; Sosnowski, M.R. Managing grapevine trunk diseases with respect to etiology and epidemiology: Current strategies and future prospects. Plant Dis. 2018, 102, 12–39. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  11. Gramaje, D.; Armengol, J. Fungal trunk pathogens in the grapevine propagation process: Potential inoculum sources, detection, identification, and management strategies. Plant Dis. 2011, 95, 1040–1055. [Google Scholar] [CrossRef] [Green Version]
  12. Gramaje, D.; Mostert, L.; Groenewald, J.Z.; Crous, P.W. Phaeoacremonium: From esca disease to phaeohyphomycosis. Fungal Biol. 2015, 119, 759–783. [Google Scholar] [CrossRef]
  13. Halleen, F.; Mostert, L.; Crous, P.W. Pathogenicity testing of lesser-known vascular fungi of grapevines. Australas. Plant Pathol. 2007, 36, 277–285. [Google Scholar] [CrossRef]
  14. Agustí-Brisach, C.; Armengol, J. Black-foot disease of grapevine: An update on taxonomy, epidemiology and management strategies. Phytopathol. Mediterr. 2013, 52, 245–261. [Google Scholar] [CrossRef]
  15. Carlucci, A.; Lops, F.; Mostert, L.; Halleen, F.; Raimondo, M.L. Occurrence fungi causing black foot on young grapevines and nursery rootstock plants in Italy. Phytopathol. Mediterr. 2017, 56, 10–39. [Google Scholar] [CrossRef]
  16. Lombard, L.; Van der Merwe, N.A.; Groenewald, J.Z.; Crous, P.W. Lineages in Nectriaceae: Re-evaluating the generic status of Ilyonectria and allied genera. Phytopathol. Mediterr. 2014, 53, 515–532. [Google Scholar]
  17. Baumgartner, K.; Fujiyoshi, P.T.; Travadon, R.; Castlebury, L.A.; Wilcox, W.F.; Rolshausen, P.E. Characterization of species of Diaporthe from wood cankers of grape in eastern North American vineyards. Plant Dis. 2013, 97, 912–920. [Google Scholar] [CrossRef] [Green Version]
  18. Úrbez-Torres, J.R.; Peduto, F.; Smith, R.J.; Gubler, W.D. Phomopsis dieback: A grapevine trunk disease caused by Phomopsis viticola in California. Plant Dis. 2013, 97, 1571–1579. [Google Scholar] [CrossRef] [Green Version]
  19. Yang, T.; Groenewald, J.Z.; Cheewangkoon, R.; Jami, F.; Abdollahzadeh, J.; Lombard, L.; Crous, P.W. Families, genera, and species of Botryosphaeriales. Fungal Biol. 2017, 121, 322–346. [Google Scholar] [CrossRef] [PubMed]
  20. Urbez-Torres, J.R. The status of Botryosphaeriaceae species infecting grapevines. Phytopathol. Mediterr. 2011, 50, 5–45. [Google Scholar] [CrossRef]
  21. Pitt, W.M.; Huang, R.; Steel, C.C.; Savocchia, S. Pathogenicity and epidemiology of Botryosphaeriaceae species isolated from grapevines in Australia. Australas. Plant Pathol. 2013, 42, 573–582. [Google Scholar] [CrossRef]
  22. Pitt, W.M.; Huang, R.; Trouillas, F.P.; Steel, C.C.; Savocchia, S. Evidence that Eutypa lata and other diatrypaceous species occur in New South Wales vineyards. Australas. Plant Pathol. 2010, 39, 97–106. [Google Scholar] [CrossRef]
  23. Trouillas, F.P.; Úrbez-Torres, J.R.; Gubler, W.D. Diversity of diatrypaceous fungi associated with grapevine canker diseases in California. Mycologia 2010, 102, 319–336. [Google Scholar] [CrossRef]
  24. Berlanas, C.; Andrés-Sodupe, M.; López-Manzanares, B.; Maldonado-González, M.M.; Gramaje, D. Effect of white mustard cover crop residue, soil chemical fumigation and Trichoderma spp. root treatment on black-foot disease control in grapevine. Pest Manag. Sci. 2018, 74, 2864–2873. [Google Scholar] [CrossRef]
  25. del Martínez-Diz, M.P.; Díaz-Losada, E.; Andrés-Sodupe, M.; Bujanda, R.; Maldonado-González, M.M.; Ojeda, S.; Yacoub, A.; Rey, P.; Gramaje, D. Field evaluation of biocontrol agents against black-foot and Petri diseases of grapevine. Pest Manag. Sci. 2020, 77, 697–708. [Google Scholar] [CrossRef] [PubMed]
  26. Van Jaarsveld, W.J.; Halleen, F.; Bester, M.C.; Pierron, R.J.G.; Stempien, E.; Mostert, L. Investigation of Trichoderma species colonization of nursery grapevines for improved management of black foot disease. Pest Manag. Sci. 2021, 77, 397–405. [Google Scholar] [CrossRef]
  27. Halleen, F.; Fourie, P.H.; Lombard, P.J. Protection of grapevine pruning wounds against Eutypa lata by biological and chemical methods. S. Afr. J. Enol. Vitic. 2010, 31, 125–132. [Google Scholar] [CrossRef] [Green Version]
  28. del Martinez-Diz, M.P.; Diaz-Losada, E.; Díaz-Fernández, Á.; Bouzas-Cid, Y.; Gramaje, D. Protection of grapevine pruning wounds against Phaeomoniella chlamydospora and Diplodia seriata by commercial biological and chemical methods. Crop Prot. 2021, 143, 105465. [Google Scholar] [CrossRef]
  29. Brader, G.; Compant, S.; Vescio, K.; Mitter, B.; Trognitz, F.; Ma, L.-J.; Sessitsch, A. Ecology and genomic insights into plant-pathogenic and -nonpathogenic endophytes. Annu. Rev. Phytopathol. 2017, 55, 61–83. [Google Scholar] [CrossRef]
  30. Keane, P.; Kerr, A. Factors affecting disease development. In Plant Pathogens and Plant Diseases; Brown, J.F., Ogle, H.J., Eds.; Rockvale: Armidale, Australia, 1997; pp. 287–298. [Google Scholar]
  31. Kredics, L.; Antal, Z.; Manczinger, L.; Szekeres, A.; Kevei, F.; Nagy, E. Influence of environmental parameters on Trichoderma strains with biocontrol potential. Food Technol. Biotechnol. 2003, 41, 37–42. [Google Scholar]
  32. Fedele, G.; Bove, F.; González-Domínguez, E.; Rossi, V. A generic model accounting for the interactions among pathogens, host plants, biocontrol agents, and the environment, with parametrization for Botrytis cinerea on grapevines. Agronomy 2020, 10, 222. [Google Scholar] [CrossRef] [Green Version]
  33. Fedele, G.; Brischetto, C.; Rossi, V. Biocontrol of Botrytis cinerea on grape berries as influenced by temperature and humidity. Front. Plant Sci. 2020, 11, 1232. [Google Scholar] [CrossRef] [PubMed]
  34. Di Lelio, I.; Coppola, M.; Comite, E.; Molisso, D.; Lorito, M.; Woo, S.L.; Pennacchio, F.; Rao, R.; Digilio, M.C. Temperature Differentially Influences the Capacity of Trichoderma Species to Induce Plant Defense Responses in Tomato Against Insect Pests. Front. Plant Sci. 2021, 12, 1–15. [Google Scholar] [CrossRef]
  35. Masson-Delmonte, V.; Zhai, P.; Pirani, A.; Connors, S.; Péan, C.; Berger, S.; Caud, N.; Chen, Y.; Goldfarb, L.; Gomis, M.; et al. IPCC 2021: Climate Change 2021: The Physical Science Basis: Contribution of Working Group I to the Sixth Assessment Report of the Intergovernmental Panel on Climate Change; Cambridge University Press: Cambridge, UK, 2021; in press. [Google Scholar]
  36. Van Leeuwen, C.; Darriet, P. The impact of climate change on viticulture and wine quality. J. Wine Econ. 2016, 11, 150–167. [Google Scholar] [CrossRef] [Green Version]
  37. Parker, A.K.; García de Cortázar-Atauri, I.; Gény, L.; Spring, J.L.; Destrac, A.; Schultz, H.; Molitor, D.; Lacombe, T.; Graça, A.; Monamy, C.; et al. Temperature-based grapevine sugar ripeness modelling for a wide range of Vitis vinifera L. cultivars. Agric. For. Meteorol. 2020, 285–286, 107902. [Google Scholar] [CrossRef]
  38. Martínez-Lüscher, J.; Sánchez-Díaz, M.; Delrot, S.; Aguirreolea, J.; Pascual, I.; Gomès, E. Ultraviolet-B alleviates the uncoupling effect of elevated CO2 and increased temperature on grape berry (Vitis vinifera cv. Tempranillo) anthocyanin and sugar accumulation. Aust. J. Grape Wine Res. 2016, 22, 87–95. [Google Scholar] [CrossRef]
  39. Gomès, É.; Maillot, P.; Duchêne, É. Molecular tools for adapting viticulture to climate change. Front. Plant Sci. 2021, 12. [Google Scholar] [CrossRef] [PubMed]
  40. Martínez-Lüscher, J.; Morales, F.; Sánchez-Díaz, M.; Delrot, S.; Aguirreolea, J.; Gomès, E.; Pascual, I. Climate change conditions (elevated CO2 and temperature) and UV-B radiation affect grapevine (Vitis vinifera cv. Tempranillo) leaf carbon assimilation, altering fruit ripening rates. Plant Sci. 2015, 236, 168–176. [Google Scholar] [CrossRef]
  41. Lecourieux, D.; Kappel, C.; Claverol, S.; Pieri, P.; Feil, R.; Lunn, J.E.; Bonneu, M.; Wang, L.; Gomès, E.; Delrot, S.; et al. Proteomic and metabolomic profiling underlines the stage- and time-dependent effects of high temperature on grape berry metabolism. J. Integr. Plant Biol. 2020, 62, 1132–1158. [Google Scholar] [CrossRef] [PubMed]
  42. Petrie, P.R.; Clingeleffer, P.R. Effects of temperature and light (before and after budburst) on inflorescence morphology and flower number of Chardonnay grapevines (Vitis vinifera L.). Aust. J. Grape Wine Res. 2005, 11, 59–65. [Google Scholar] [CrossRef]
  43. Valencia, E.; Gross, N.; Quero, J.L.; Carmona, C.P.; Ochoa, V.; Gozalo, B.; Delgado-Baquerizo, M.; Dumack, K.; Hamonts, K.; Singh, B.K.; et al. Cascading effects from plants to soil microorganisms explain how plant species richness and simulated climate change affect soil multifunctionality. Glob. Chang. Biol. 2018, 24, 5642–5654. [Google Scholar] [CrossRef] [PubMed]
  44. Campisano, A.; Albanese, D.; Yousaf, S.; Pancher, M.; Donati, C.; Pertot, I. Temperature drives the assembly of endophytic communities’ seasonal succession. Environ. Microbiol. 2017, 19, 3353–3364. [Google Scholar] [CrossRef]
  45. Delgado-Baquerizo, M.; Eldridge, D.J.; Ochoa, V.; Gozalo, B.; Singh, B.K.; Maestre, F.T. Soil microbial communities drive the resistance of ecosystem multifunctionality to global change in drylands across the globe. Ecol. Lett. 2017, 20, 1295–1305. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  46. Rodríguez-González, Á.; Carro-Huerga, G.; Mayo-Prieto, S.; Lorenzana, A.; Gutiérrez, S.; Peláez, H.J.; Casquero, P.A. Investigations of Trichoderma spp. and Beauveria bassiana as biological control agent for Xylotrechus arvicola, a major insect pest in Spanish vineyards. J. Econ. Entomol. 2018, 111, 2585–2591. [Google Scholar] [CrossRef] [Green Version]
  47. Carro-Huerga, G.; Compant, S.; Gorfer, M.; Cardoza, R.E.; Schmoll, M.; Gutiérrez, S.; Casquero, P.A. Colonization of Vitis vinifera L. by the endophyte Trichoderma sp. strain T154: Biocontrol activity against Phaeoacremonium minimum. Front. Plant Sci. 2020, 11, 1170. [Google Scholar] [CrossRef] [PubMed]
  48. Kubicek, C.P.; Steindorff, A.S.; Chenthamara, K.; Manganiello, G.; Henrissat, B.; Zhang, J.; Cai, F.; Kopchinskiy, A.G.; Kubicek, E.M.; Kuo, A.; et al. Evolution and comparative genomics of the most common Trichoderma species. BMC Genomics 2019, 20, 1–24. [Google Scholar] [CrossRef] [Green Version]
  49. Martín, M.T.; Cobos, R.; Martin, M.T.; Cobos, R. Identification of fungi associated with grapevine decline in Castilla y León (Spain). Phytopathol. Mediterr. 2007, 46, 18–25. [Google Scholar] [CrossRef]
  50. Qiu, Y.; Steel, C.C.; Ash, G.J.; Savocchia, S. Effects of temperature and water stress on the virulence of Botryosphaeriaceae spp. causing dieback of grapevines and their predicted distribution using CLIMEX in Australia. Acta Hortic. 2016, 1115, 171–181. [Google Scholar] [CrossRef]
  51. Bellée, A.; Comont, G.; Nivault, A.; Abou-Mansour, E.; Coppin, C.; Dufour, M.C.; Corio-Costet, M.F. Life traits of four Botryosphaeriaceae species and molecular responses of different grapevine cultivars or hybrids. Plant Pathol. 2017, 66, 763–776. [Google Scholar] [CrossRef] [Green Version]
  52. Valtaud, C.; Larignon, P.; Roblin, G.; Fleurat-Lessard, P. Developmental and ultrastructural features of Phaeomoniella chlamydospora and Phaeoacremonium aleophilum in relation to xylem degradation in esca disease of the grapevine. J. Plant Pathol. 2009, 91, 37–51. [Google Scholar] [CrossRef]
  53. Mutawila, C.; Halleen, F.; Mostert, L. Optimisation of time of application of Trichoderma biocontrol agents for protection of grapevine pruning wounds. Aust. J. Grape Wine Res. 2016, 22, 279–287. [Google Scholar] [CrossRef]
  54. Rifai, M.A. A revision of the genus Trichoderma. Mycol. Pap. 1969, 116, 1–56. [Google Scholar]
  55. Butt, T.M.; Jackson, C.; Magan, N. Fungi as Biocontrol Agents Progress, Problems and Potential; CABI: Wallingford, UK, 2001; pp. 1–8. [Google Scholar]
  56. Singh, A.; Shahid, M.; Srivastava, M.; Pandey, S.; Sharma, A.; Kumar, V. Optimal physical parameters for growth of Trichoderma species at varying pH, temperature and agitation. Virol. Micol. 2015, 3, 1–8. [Google Scholar] [CrossRef]
  57. Kubicek, C.P.; Harman, G.E. Basic biology, taxonomy and genetics. In Trichoderma and Gliocladium; CRC Press: Abingdon, UK, 1998; Volume 1, pp. 57–69. ISBN 9780748405725. [Google Scholar]
  58. Rinu, K.; Sati, P.; Pandey, A. Trichoderma gamsii (NFCCI 2177): A newly isolated endophytic, psychrotolerant, plant growth promoting, and antagonistic fungal strain. J. Basic Microbiol. 2014, 54, 408–417. [Google Scholar] [CrossRef] [PubMed]
  59. Shanmugam, V.; Chugh, P.; Sharma, P. Cold-tolerant Trichoderma species for the management of Fusarium wilt of tomato plants. Ann. Microbiol. 2015, 65, 543–551. [Google Scholar] [CrossRef]
  60. Harman, G.E.; Kubicek, C.P. Enzymes, biological control and commercial applications. In Trichoderma and Gliocladium; CRC Press: Abingdon, UK, 1998; Volume 2, ISBN 9780748405725. [Google Scholar]
  61. Antal, Z.; Manczinger, L.; Szakacs, G.; Tengerdy, R.P.; Ferenczy, L. Colony growth, in vitro antagonism and secretion of extracellular enzymes in cold-tolerant strains of Trichoderma species. Mycol. Res. 2000, 104, 545–549. [Google Scholar] [CrossRef]
  62. Singh, A.; Srivastava, S.; Singh, H.B. Effect of substrates on growth and shelf life of Trichoderma harzianum and its use in biocontrol of diseases. Bioresour. Technol. 2007, 98, 470–473. [Google Scholar] [CrossRef]
  63. Danielson, R.M.; Davey, C.B. Non nutritional factors affecting the growth of Trichoderma in culture. Soil Biol. Biochem. 1973, 5, 495–504. [Google Scholar] [CrossRef]
  64. Danielson, R.M.; Davey, C.B. The abundance of Trichoderma propagules and the distribution of species in forest soils. Soil Biol. Biochem. 1973, 5, 485–494. [Google Scholar] [CrossRef]
  65. Fraga, H.; Malheiro, A.C.; Moutinho-Pereira, J.; Santos, J.A. An overview of climate change impacts on European viticulture. Food Energy Secur. 2012, 1, 94–110. [Google Scholar] [CrossRef]
  66. Jaklitsch, W.M.; Samuels, G.J.; Dodd, S.L.; Lu, B.S.; Druzhinina, I.S. Hypocrea rufa/Trichoderma viride: A reassessment, and description of five closely related species with and without warted conidia. Stud. Mycol. 2006, 55, 135–177. [Google Scholar] [CrossRef] [Green Version]
  67. Anees, M.; Tronsmo, A.; Edel-Hermann, V.; Hjeljord, L.G.; Héraud, C.; Steinberg, C. Characterization of field isolates of Trichoderma antagonistic against Rhizoctonia solani. Fungal Biol. 2010, 114, 691–701. [Google Scholar] [CrossRef]
  68. Druzhinina, I.S.; Kubicek, C.P.; Komo-Zelazowska, M.; Mulaw, T.B.; Bissett, J. The Trichoderma harzianum demon: Complex speciation history resulting in coexistence of hypothetical biological species, recent agamospecies and numerous relict lineages. BMC Evol. Biol. 2010, 10, 1–14. [Google Scholar] [CrossRef] [Green Version]
  69. Luque, J.; Elena, G.; Garcia-Figueres, F.; Reyes, J.; Barrios, G.; Legorburu, F.J. Natural infections of pruning wounds by fungal trunk pathogens in mature grapevines in Catalonia (Northeast Spain). Aust. J. Grape Wine Res. 2014, 20, 134–143. [Google Scholar] [CrossRef]
  70. Martínez-Diz, M. del P.; Eichmeier, A.; Spetik, M.; Bujanda, R.; Díaz-Fernández, Á.; Díaz-Losada, E.; Gramaje, D. Grapevine pruning time affects natural wound colonization by wood-invading fungi. Fungal Ecol. 2020, 48, 100994. [Google Scholar] [CrossRef]
  71. Carro-Huerga, G.; Mayo-Prieto, S.; Rodríguez-González, Á.; González-López, Ó.; Gutierrez, S.; Casquero, P.A. Influence of fungicide application and vine age on Trichoderma diversity as source of biological control agents. Agronomy 2021, 11, 446. [Google Scholar] [CrossRef]
  72. Mutawila, C.; Fourie, P.H.; Halleen, F.; Mostert, L. Grapevine cultivar variation to pruning wound protection by Trichoderma species against trunk pathogens. Phytopathol. Mediterr. 2011, 50, 264–276. [Google Scholar] [CrossRef]
  73. Mutawila, C.; Halleen, F.; Mostert, L. Development of benzimidazole resistant Trichoderma strains for the integration of chemical and biocontrol methods of grapevine pruning wound protection. BioControl 2015, 60, 387–399. [Google Scholar] [CrossRef]
  74. Kotze, C.; Van Niekerk, J.; Mostert, L.; Halleen, F.; Fourie, P.; Van Niekerk, J.; Mostert, L.; Halleen, F.; Fourie, P. Evaluation of biocontrol agents for grapevine pruning wound protection against trunk pathogen infection. Phytopathol. Mediterr. 2011, 50, 247–263. [Google Scholar] [CrossRef]
  75. Eskalen, A.; Feliciano, A.J.; Gubler, W.D. Susceptibility of grapevine pruning wounds and symptom development in response to infection by Phaeoacremonium aleophilum and Phaeomoniella chlamydospora. Plant Dis. 2007, 91, 1100–1104. [Google Scholar] [CrossRef] [Green Version]
  76. John, S.; Wicks, T.J.; Hunt, J.S.; Scott, E.S. Colonisation of grapevine wood by Trichoderma harzianum and Eutypa lata. Aust. J. Grape Wine Res. 2008, 14, 18–24. [Google Scholar] [CrossRef]
  77. Prozil, S.O.; Evtuguin, D.V.; Lopes, L.P.C. Chemical composition of grape stalks of Vitis vinifera L. from red grape pomaces. Ind. Crops Prod. 2012, 35, 178–184. [Google Scholar] [CrossRef]
  78. Hjelford, L.G.; Stensvand, A.; Tronsmo, A. Effect of temperature and nutrient stress on the capacity of commercial Trichoderma products to control Botrytis cinerea and Mucor piriformis in greenhouse strawberries. Biol. Control 2000, 19, 149–160. [Google Scholar] [CrossRef]
  79. Mayo-Prieto, S.; Campelo, M.P.; Lorenzana, A.; Rodríguez-González, A.; Reinoso, B.; Gutiérrez, S.; Casquero, P.A. Antifungal activity and bean growth promotion of Trichoderma strains isolated from seed vs soil. Eur. J. Plant Pathol. 2020, 158, 817–828. [Google Scholar] [CrossRef]
  80. John, S.; Wicks, T.J.; Hunt, J.S.; Lorimer, M.F.; Oakey, H.; Scott, E.S. Protection of grapevine pruning wounds from infection by Eutypa lata. Aust. J. Grape Wine Res. 2005, 14, 134–142. [Google Scholar] [CrossRef]
  81. Matese, A.; Di Gennaro, S.F. Technology in precision viticulture: A state of the art review. Int. J. Wine Res. 2015, 7, 69–81. [Google Scholar] [CrossRef] [Green Version]
  82. Pertot, I.; Prodorutti, D.; Colombini, A.; Pasini, L. Trichoderma atroviride SC1 prevents Phaeomoniella chlamydospora and Phaeoacremonium aleophilum infection of grapevine plants during the grafting process in nurseries. BioControl 2016, 61, 257–267. [Google Scholar] [CrossRef]
  83. Di Marco, S.; Osti, F.; Cesari, A. Experiments on the control of esca by Trichoderma. Phytopathol. Mediterr. 2004, 43, 108–115. [Google Scholar] [CrossRef]
  84. Contreras-Cornejo, H.A.; Macías-Rodríguez, L.; Cortés-Penagos, C.; López-Bucio, J. Trichoderma virens, a plant beneficial fungus, enhances biomass production and promotes lateral root growth through an auxin-dependent mechanism in arabidopsis. Plant Physiol. 2009, 149, 1579–1592. [Google Scholar] [CrossRef] [Green Version]
  85. Karimi, K.; Babai Ahari, A.; Arzanlou, M.; Amini, J.; Pertot, I. Comparison of indigenous Trichoderma spp. strains to a foreign commercial strain in terms of biocontrol efficacy against Colletotrichum nymphaeae and related biological features. J. Plant Dis. Prot. 2017, 124, 453–466. [Google Scholar] [CrossRef]
  86. Mayo, S.; Gutiérrez, S.; Malmierca, M.G.; Lorenzana, A.; Campelo, M.P.; Hermosa, R.; Casquero, P.A. Influence of Rhizoctonia solani and Trichoderma spp. in growth of bean (Phaseolus vulgaris L.) and in the induction of plant defense-related genes. Front. Plant Sci. 2015, 6, 685. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  87. Álvarez-Pérez, J.M.; González-García, S.; Cobos, R.; Olego, M.Á.; Ibañez, A.; Díez-Galán, A.; Garzón-Jimeno, E.; Coque, J.J.R. Use of endophytic and rhizosphere actinobacteria from grapevine plants to reduce nursery fungal graft infections that lead to young grapevine decline. Appl. Environ. Microbiol. 2017, 83, e01564-17. [Google Scholar] [CrossRef] [PubMed] [Green Version]
Agronomy 11 01771 g001 550
Figure 1. Growth of the Trichoderma strains T154 and T071 after 7 days at 4 °C, 15 °C, 25 °C, and 35 °C.
Figure 1. Growth of the Trichoderma strains T154 and T071 after 7 days at 4 °C, 15 °C, 25 °C, and 35 °C.
Agronomy 11 01771 g001
Agronomy 11 01771 g002 550
Figure 2. Evaluation of the growth of the Trichoderma strains at a different range of temperature (4 °C, 15 °C, 25 °C, and 35 °C). Values in each graph followed by the same letter do not differ significantly (p = 0.05).
Figure 2. Evaluation of the growth of the Trichoderma strains at a different range of temperature (4 °C, 15 °C, 25 °C, and 35 °C). Values in each graph followed by the same letter do not differ significantly (p = 0.05).
Agronomy 11 01771 g002
Agronomy 11 01771 g003 550
Figure 3. Spore production at 25°C after 7 days in a PDA plate. Values followed by the same letter do not differ significantly (p = 0.05).
Figure 3. Spore production at 25°C after 7 days in a PDA plate. Values followed by the same letter do not differ significantly (p = 0.05).
Agronomy 11 01771 g003
Agronomy 11 01771 g004 550
Figure 4. Growth rate of the different Trichoderma strains analyzed in this work from two spore concentrations at 15 °C. Values followed by the same letter do not differ significantly (p = 0.05).
Figure 4. Growth rate of the different Trichoderma strains analyzed in this work from two spore concentrations at 15 °C. Values followed by the same letter do not differ significantly (p = 0.05).
Agronomy 11 01771 g004
Agronomy 11 01771 g005 550
Figure 5. (A) Inoculation point. (B) Points of evaluation after inoculation.
Figure 5. (A) Inoculation point. (B) Points of evaluation after inoculation.
Agronomy 11 01771 g005
Agronomy 11 01771 g006 550
Figure 6. (A) Average temperature of each week. (B) Average relative humidity of each week.
Figure 6. (A) Average temperature of each week. (B) Average relative humidity of each week.
Agronomy 11 01771 g006
Agronomy 11 01771 g007 550
Figure 7. Percentage of re-isolation of pruning wounds after 12 weeks applying Trichoderma strain from T071 and T154. Values followed by the same letter do not differ significantly (p = 0.05).
Figure 7. Percentage of re-isolation of pruning wounds after 12 weeks applying Trichoderma strain from T071 and T154. Values followed by the same letter do not differ significantly (p = 0.05).
Agronomy 11 01771 g007
Table
Table 1. Trichoderma isolates used in this study.
Table 1. Trichoderma isolates used in this study.
Number IsolateReference 1OriginMain Mechanism of Biocontrol
T065This studyVineyard soil-
T071T. gamsii (Section Trichoderma) [46].Vineyard soilMycoparasistim in insects
T154Trichoderma spp. (Clade Harzianum/Virens) [47].Grapevine barkMycoparasistim of fungi and niche exclusion
T214This studyGrapevine bark-
1 Section/clade identification performed according to Kubicek et al., 2019 [48].
Table
Table 2. Temperature growth range of the main fungi associated with GTDs in Castilla y León region.
Table 2. Temperature growth range of the main fungi associated with GTDs in Castilla y León region.
Most Frequent Fungi Associated with GTDs Isolated in Castilla y León Region 1Minimal Temperature for Mycelial Growth (°C)Maximum Temperature for Mycelial Growth (°C)Reference of Temperature Conditions
Botryosphaeria dothidea (Moug.) Ces. and De Not435[50]
Botryosphaeria parva Pennycook and Samuels435[50]
Botryosphaeria stevensii Shoemaker1533[51]
Diplodia seriata De Not435[50]
Phaeoacremonium minimum (Tul. and C. Tul.) Gramaje, L. Mostert and Crous1030[52]
Phaeomoniella chlamydospora (W. Gams, Crous, M.J. Wingf. and Mugnai) Crous and W. Gams1035[52]
1 species described according to Cobos et al., 2007 [49].
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Carro-Huerga, G.; Mayo-Prieto, S.; Rodríguez-González, Á.; Álvarez-García, S.; Gutiérrez, S.; Casquero, P.A. The Influence of Temperature on the Growth, Sporulation, Colonization, and Survival of Trichoderma spp. in Grapevine Pruning Wounds. Agronomy 2021, 11, 1771. https://doi.org/10.3390/agronomy11091771

AMA Style

Carro-Huerga G, Mayo-Prieto S, Rodríguez-González Á, Álvarez-García S, Gutiérrez S, Casquero PA. The Influence of Temperature on the Growth, Sporulation, Colonization, and Survival of Trichoderma spp. in Grapevine Pruning Wounds. Agronomy. 2021; 11(9):1771. https://doi.org/10.3390/agronomy11091771

Chicago/Turabian Style

Carro-Huerga, Guzmán, Sara Mayo-Prieto, Álvaro Rodríguez-González, Samuel Álvarez-García, Santiago Gutiérrez, and Pedro A. Casquero. 2021. "The Influence of Temperature on the Growth, Sporulation, Colonization, and Survival of Trichoderma spp. in Grapevine Pruning Wounds" Agronomy 11, no. 9: 1771. https://doi.org/10.3390/agronomy11091771

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop