Heritage grapevines as sources of biological control agents for Botryosphaeria dieback pathogens

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Among the fungi associated with Botryosphaeria dieback, Lasiodiplodia and Neofusicoccum contain the most virulent species identified in different countries, including the United States of America (Úrbez-Torres and Gubler, 2009), Iraq (Abdullah et al., 2012) and Mexico (Rangel-Montoya et al., 2021).Lasiodiplodia spp.have white colonies in culture, that later become dark gray with abundant mycelium, and their conidia can be aseptate hyaline or septate pigmented, with longitudinal striations.Neofusicoccum spp.have initially white colonies, and some species as N. luteum and N. australe produce yellow pigments.Their abundant aerial mycelium later turns gray, and conidia are hyaline, unicellular, and aseptate, with a subtruncate bases (Zhang et al., 2021).
Management of GTDs is complex, and no curative treatments are known to date.Therefore, strategies commonly focus on implementation of cultural and preventive treatment measures, with each depending on plant damage, the pathogens involved, and the geographic region (Gramaje et al., 2018).These include the protection of pruning wounds with fungicides or biological control agents (BCAs), and curative surgery (Mondello et al., 2018).
The search for endophytic microorganisms for biological control of GTDs pathogens has increased, due to their potential to have antagonistic activities against different species of fungi and, in some cases, to also promote plant growth (Dini-Andreote, 2020; Jacob et al., 2020).Different endophytic microorganisms have been reported as antagonists of GTDs pathogens.These include fungi (Aspergillus, Chaetomium, Clonostachys, Cladosporium, Epicoccum, Fusarium, and Trichoderma), which have shown antagonistic in vitro activity against Diplodia and Neofusicoccum spp.(Almeida et al., 2020;Silva-Valderrama et al., 2021).As well, bacteria (Bacillus, Burkholderia, Paenibacillus, Pseudomonas, and Streptomyces) have been recognized as antagonists of Diplodia, Lasiodiplodia, and Neofusiccocum (Haidar et al., 2016).Among the organisms with potential as BCAs, Bacillus and Trichoderma show in vitro antagonistic activity through different mechanisms of action, including competition for space, production of volatile and non-volatile compounds, or mycoparasitism.In greenhouse and field trials, these organisms have shown efficacy for control of GTDs fungi when applied to grapevine pruning wounds (Almeida et al., 2020).
In Mexico, the Spanish introduced grapevine (Vitis vinifera) in the 16th century, and Jesuit and Dominican friars disseminated its cultivation (Crowley, 1989).'Listan Prieto', now known as 'Mission', was introduced in Mexico and in the United States of America (Walker et al., 2019).In the Baja California peninsula, the first grapevines were established in the San Francisco Javier mission, located in Loreto (Magoni, 2009).From then on, grapevine cultivation continued to expand.Currently in Baja California, 'Mission' grapevines are over 40 years old, and have been propagated from the first vines introduced in the state by the Jesuit missionaries, and these plants are considered to be heritage grapevines.Approx.38 ha of this variety are grown with minimal management in Baja California, with yields of approx.3.5 tons ha -1 (SEFOA, 2011;Andrade et al., 2013).
Different reports have shown that plants growing in arid conditions associate with microorganisms (bacteria and fungi).These can enhance plant drought resistance through various mechanisms, including improved water and nutrient absorption by inducing changes in root morphology, protecting against oxidative damage, regulating phytohormone levels, and suppressing phytopathogens such as those responsible of GTDs (Poudel et al., 2021;Riseh et al., 2021).
Considering the time since they were established and the complex climatic conditions in which the 'Mission' grapevines have been planted (dry to very dry climate with average rainfall of 200 mm p.a. (INEGI, 2017), it is likely that these plants maintain associations with beneficial microorganisms which allow them to survive in the adverse conditions and resist plant pathogens.Therefore, the objective of the present study was to obtain endophytic bacteria and fungi from the cultivar 'Mission' with the potential as BCAs of Botryosphaeria dieback fungi, thus providing sustainable alternatives for the control of this disease in commercial vineyards of Baja California.

Sampling and isolation of microorganisms from heritage grapevines
Microorganisms were isolated from lignified 1-yearold branches of heritage grapevines cultivar 'Mission', growing in local vineyards in the Guadalupe valley (31.994722, -116.683896) and Ejido Uruapan (31.628436, -116-434295), Baja California.Small tissue fragments were cut from each branch sample, and after bark removal, these were surface sterilized by flaming (Rangel-Montoya et al., 2021).Subsequently, for bacteria isolations, branch fragments were transferred to plates containing LB Agar (ATCC media No.1065), YPD Agar (1245) or King's medium B Agar (1213), or PY medium (tryptone 5.0 g, yeast extract 3.0 g, CaCl 2 0.9 g, pH 6.8), supplemented with cycloheximide (final concentration 100 μg mL -1 ).For fungal isolations, tissue fragments were inserted in plates containing Potato Dextrose Agar (PDA), or water agar supplemented with chloramphenicol (final concentration 25 µg mL -1 ).The isolation plates were incubated at 30°C until microorganism growth was observed, and the resulting bacterial and fungal colonies were recovered and subcultured to obtain pure cultures.Fungal strains were preserved at 4°C in 20% glycerol, and bacteria strains in 35% glycerol solution at -20ºC.Additionally, Bacillus sp.rbES015 and 35 strains of Trichoderma were obtained from the collection of the Phytopathology Laboratory of CICESE.

Screening for antifungal activity
The GTD fungi Lasiodiplodia brasiliensis MXBCL28 (Rangel-Montoya et al., 2021) and Neofusicoccum parvum 14P4MX (Rangel-Montoya, 2021) were used to test the biological control potential of the obtained fungal and bacterial isolates.Using a flame-sterilized 7 mm cork borer, an agar plug with mycelium was obtained from a 4-day-old culture, from each fungus grown on PDA.The plug was then placed on the centre of a fresh PDA plate, and incubated at 25°C.When the fungus colony reached 1 cm diam.5 µL of four different potential BCA bacterial cultures or one mycelium plug from potential fungal BCAs, were inoculated at the edges, as described by Guevara-Avendaño et al. (2018).Plates with only GTD fungi were used as experimental controls.These assays were each carried out in triplicate.After 7 d incubation at 30°C, the inhibition of radial growth of L. brasiliensis mycelium was assessed.In total, 135 bacterial and 39 fungal isolates were screened in these assays.
After discarding isolates with low or no inhibition activity, quantitative fungal inhibition assays were carried out for 58 strains (21 bacteria and 37 fungi), as described for the qualitative assays (above).For each of these quantitative assays, the L. brasiliensis mycelium plug was placed at the edge of the Petri plate, and only one bacterial or fungi strain was inoculated directly opposed to it.The following formula (Méndez-Bravo et al., 2018) was used to calculate the percentage of inhibition of mycelial growth: %inhibition = [(R-r)/R] × 100, where R is the colony radius of the pathogenic fungus growing alone in the control plates, and r is the colony radius of the fungus growing in the plate in confrontation with a tested isolate.All in vitro antagonistic assays were performed in triplicate.
These procedures allowed selection of the most promising BCAs for used in the experiments described below.

Evaluation of antagonistic effects by volatile organic compounds
Antagonistic effects of volatile organic compounds produced by eleven selected strains (four bacteria and seven fungi) were evaluated against L. brasiliensis and N. parvum, using the two sealed base plate method of Rangel-Montoya et al. (2022).For each potential fungal BCA isolate, a mycelial plug was placed in the centre of a PDA plate, and for each bacterium, 20 µL of culture was spread in a PY plate.The lid of each plate was the replaced by a second PDA plate with a mycelial plug of the pathogen at the centre.The two plates were sealed with tape and incubated at 30°C for 4 d, with mycelium growth assessed every 24 h.As experimental controls, non-inoculated PDA or PY plates were used as the covering plate.

Evaluation of the antagonistic effects by diffusible organic compounds
The antagonistic activity of diffusible organic compounds produced by eleven selected BCAs strains was evaluated against L. brasiliensis and N. parvum.For bacteria, 5 mL of liquid PY medium was inoculated with a single colony, and then incubated at 30°C and 110 rpm in a shaker incubator.After 7 d, cultures were each centrifuged at 10,000 rpm for 20 min, and the resulting supernatant was filter-sterilized using a 20 µm syringe filter.PDA plates containing 15% (v/v) of sterile bacterial supernatant were then prepared, and a mycelial plug of each pathogen was placed in the centre of each test plate (Salvatierra-Martinez et al., 2018).The plates were then incubated at 30°C for 4 d, registering mycelium growth every 24 h.For evaluation of fungal isolates, cellulose membrane assays were used (Mayo-Prieto et al., 2020).A mycelial plug disc of each fungus was inoculated in the centre of each PDA plate.The plug was then covered with a sterile cellulose membrane, and the plates were incubated at 30°C for 48 h.The membrane with the mycelial growth was then removed, and a mycelial plug of the pathogen was placed in the centre of the plate.Plates were incubated at 30°C for 4 d, and mycelium growth was assessed every 24 h.

Evaluation of mycoparasitism activity
Mycoparasitism activity of seven selected Trichoderma isolates was assessed against L. brasiliensis and N. parvum, using the pre-colonized plate method described by Bailey et al., (2008).A mycelial plug of each pathogenic fungus was inoculated at the edge of a PDA plate, and after 5 d incubation at 30°C, an agar strip (4.0 × 0.5 cm) from a colony of a Trichoderma isolate was placed at the opposite side.The plates were incubated for 28 d at 30°C in darkness.Ten mycelial plugs were then collected from each plate in a straight line beginning near the agar strip and extending towards the opposite edge of the plate.The mycelial plugs were then inoculated into PDA plates and incubated for 24 h at 30°C in darkness, followed by 5 d incubation under white light at room temperature.As experimental controls, cultures of L. brasiliensis and N. parvum were used, grown without Trichoderma and maintained under the same conditions.Mycoparasitism was determined by assessing the presence of Trichoderma and the mycoparasitic fungi in the ten mycelial plugs collected.Micoparasitic activity was also assessed under a microscope.For each of these assessments, a dual culture assay was performed in water agar plates, and after 3 d incubation at 30°C, a fragment of agar was cut from the centre of the plate.The obtained samples were observed with inverted microscope (Zeiss Axiovert 200), and the obtained images were analyzed using Zeiss AxioVision SE64, Rel.4.9.1 software.

In vitro screening for plant growth promoting traits
Coluorimetric tests were carried out to determine plant growth promotion by the potential BCA bacteria and fungi strains.For these assays, the strains were recovered from glycerol stock cultures, and bacteria were inoculated into PY liquid medium, and fungi onto PDA medium.Bacterial cultures were incubated for 2 d at 30°C and 100 rpm in a shaker incubator.Fungi were incubated at 30°C for 7 d, Coluorimetric tests were carried out in triplicate, in 35 mm diam.Petri dishes for solid media or 10 mL capacity tubes for liquid media, as described in the sections below.Petri plates or tubes with the corresponding media but without BCAs were used as experimental controls.

Mineral solubilization assays
Some microorganisms are capable of hydrolyzing organic and inorganic insoluble mineral compounds to soluble forms, that can be assimilated by plants, acting as biofertilizers or plant grow-promoters.Phosphate, potassium, and zinc solubilization assays were performed for the potential BCAs.In each case, 5 µL of bacterium culture or a 7 mm diam.mycelial plug of fungus were inoculated at the centre of each assay plate.
Inorganic potassium solubilization was determined on modified Pikovskaya agar (Pikovskaya, 1948), using KNO 3 and bromocresol green.After inoculation, Petri plates were incubated for 72 h at 30°C.Colour change from blue to yellow indicated a positive potassium solubilizing strain.
Zinc solubilization was assessed using zinc-supplemented Pikovskaya medium complemented with 1.2 g L -1 ZnO and bromothymol blue.Inoculated Petri dishes were incubated in the dark for 72 h at 30°C.Colour change from blue to yellow indicated a positive zinc solubilizing strain (Bapiri et al., 2012).

Indole Acetic Acid (AIA) production assays
Indole acetic acid (IAA) is one of the most physiologically active auxins.It induces production of long roots and root hairs, and lateral roots, which are involved in nutrient uptake by plants (Datta and Basu, 2000).For determination of IAA production by bacterial strains, 5 µL of 1-d-old cultures were reinoculated in 96 well micro-plates, with 200 µL PY liquid medium supplemented with tryptophan to a final concentration of 500 µg mL -1 .Microplates were incubated for 48 h at 30°C and 110 rpm in a shaker incubator.Subsequently, 100 µL of Salkowski reagent (50 mL, 35% HClO 4, 1 mL 0.5 M FeCl 3 ) (Ahmad et al., 2008) were then added per well, and the microplates were each covered with aluminum foil and incubated for 30 min.For each fungal strain, three 7 mm diam.mycelial plugs were inoculated into 5 mL of PDB medium, and these cultures were incubated for 7 d at 30°C and 110 rpm in a shaker incubator.Two hundred µL of the culture were then placed in 96 well microplates, and 100 µL of Salkowski reagent were immediately added.Microplates were each covered with aluminum foil and incubated for 30 min.For the bacteria and fungi, change of colour to pink indicated a positive result.

Hydrogen cyanide production assays
Hydrogen cyanide (HCN) is produced by some BCAs, and its toxicity to phytopathogens makes the BCAs suitable for biocontrol.After inoculating the bacteria onto solid PY and the fungi onto PDA in Petri plates, a filter paper moistened with a solution of 0.5% sodium carbonate in 0.5% picric acid (Ahmad et al., 2008) was fixed to each Petri plate cover.The plates were subsequently sealed with parafilm and incubated in dark at 30°C for 4 d.Development of orange-red colour indicated positive hydrogen cyanide producer strains.

Siderophore production assays
Siderophores are competitive traits used for BCAs to sequester iron, depriving pathogens of this element required for their growth and pathogenesis.To test for siderophore production, chrome azurol S agar (CAS) medium (Schwyn and Neilands, 1987) was prepared as described by Lynne et al. (2011).In the centre of each assay plate was inoculated 5 µL of bacterial culture or a 7 mm diam.mycelial plug of fungus.The Petri dishes were incubated in the dark for 96 h at 30°C.Colour changes from blue to yellow indicated siderophore producing strains.
All obtained PCR products were verified by electrophoresis on 1% agarose gels, purified using the GenElute PCR Clean-Up Kit (Sigma-Aldrich), and sent to Eton Bioscience Inc. for sequencing.
The resulting sequences were aligned using MEGA XI (Kumar et al., 2018), with the multiple alignment program MUSCLE. the bacteria sequences were blasted against the GenBank 16S Ribosomal RNA sequences database (Table 1), and the Trichoderma spp.sequences were compared with the GenBank elongation factor 1α gene sequences database (Table 2), and the clos-est matches were used to construct each alignment.A Maximum-Parsimony method was used with Bootstrap values based on 1,000 replicates.New sequences were deposited in the GenBank (Tables 1 and 2).

Greenhouse biocontrol assays of Lasiodiplodia brasiliensis
Grapevine plants (Vitis vinifera 'Cabernet Sauvignon') obtained from 1-year-old cuttings were used to determine the biocontrol activity of selected bacterial and fungal strains and a rhizosphere strain rbES015 obtained in a previous study (Delgado-Ramírez et al., 2021).Grapevine shoots were submerged in a 3 g L -1 solution of rooting agent ROOTEX (Cosmocel SA), and were then planted in tubs containing Cosmopeat substrate (Cosmocel SA).After 45 d, the plants were transplanted into 3.78 L plastic pots.Two weeks after transplanting, 50 mL of a solution (1 × 10 6 CFU) of each potential beneficial microorganism was applied at the base of the plant stem, followed by a second application 7 d later.Control treatments were inoculated with sterile water.For each tested isolate, ten replicates were used.Immediately after the second application of potential BCA, inoculations of the plants with L. brasiliensis were carried out through mechanical wounds in the woody tissues, each made with a drill bit (2 mm diam.), followed by insertion of a mycelium plug inside each hole.After inoculation, the wounds were each covered with parafilm.Plugs of sterile PDA were used as experimental control inoculations.The plants were then kept under greenhouse conditions for 60 d, and necrotic lesions generated in the stems were measured.Attempts were also made to recover the inoculated microorganisms.

Vineyard biocontrol assays of Lasiodiplodia brasiliensis
A field biocontrol trial was carried out in a 2-yearold 'Chenin Blanc' vineyard, in Ejido el Porvenir, Baja Isolates from this study are highlighted in bold font.
California.Fifty plants, which did not show symptoms associated with wood diseases, were chosen per row on five vineyard rows, leaving an interval of three to five plants between each selected vine.The experimental design was completely randomized with ten grapevines per treatment.Putative BCAs evaluated included five bacteria (BEVP01BCMX, BEVP02BCMX, BEV-P26BCMX, BEVP31BCMX, and rbES015) and six fungi (T06BCMX, T11BCMX, T15BCMX, T17BCMX, T20BC-MX, and EF11BCMX).In each selected plant, a pruning cut was made in a woody branch, and 10 µL of a 1 × 10 6 CFU suspension of the selected biocontrol organism were inoculated, and 20 plants were treated with each isolate.Five days later, a second inoculation of the biocontrol agent was made in the same wound.One hour later, 10 µL of a 1x10 5 suspension of fragmented mycelium of L. brasiliensis was applied to ten of the plants.
Negative controls were inoculated only with sterile distilled water.The inoculated branches were each sealed with parafilm and then covered with a paper bag (Figure S1).One month later, the treated branches were cut, the length of the lesions produced by L. brasiliensis was measured, and a tissue fragment from each branch was inoculated onto PDA to assess if the pathogen and the inoculated BCA was present.

Statistical analyses
Data obtained from the greenhouse and vineyard biocontrol experiments were analyzed using one-way ANOVA, with post-hoc LSD analysis, and an α < 0.05 test for statistical significance, using the STATISTICA 8.0 package.Isolates from this study are highlighted in bold font.

Isolation, screening and molecular identification of microorganisms from heritage grapevines
A total of 135 isolates of bacteria from the heritage grapevine tissues were characterized by morphological characteristics as Bacillus, Paenibacillus and Pseudomonas.Isolates of fungi included two Trichoderma spp., and Alternaria, Chaetomium, Sordaria and Diplodia spp.Given the small number of potential beneficial fungal isolates recovered, 35 uncharacterized Trichoderma strains from our laboratory collection were included in this study.
From the 172 evaluated strains, 37 fungal and 21 bacterial isolates showed antagonistic activity in the qualitative antagonism assays (Figure 1).Quantitative dual culture assays and screening for plant growth promotion traits were performed only for those 58 isolates.Results showed that mean inhibition proportions for these BCAs against L. brasiliensis were between 3.4% to 52.8%, and that the isolates had different plant growth promotion characteristics (Table S1).

Evaluation of antifungal effect of volatile and diffusible organic compounds
The eleven isolates were further screened for the antifungal activity from diffusible and volatile organic compounds.None of the assessed Trichoderma or Bacillus isolates produced volatile organic compounds with suppressive effects on L. brasiliensis (Figure 2; Table 4).However, all the Bacillus isolates affected growth of N. parvum, with mean inhibition percentages ranging from 22.6% to 34.0%.Isolate BEVP31BCMX gave the greatest inhibition (Figure 2; Table 4).In contrast, the 11 isolates affected the growth of both pathogenic fungi by the production of diffusible organic compounds.The Bacillus isolates gave mean inhibition percentages from 40.4% to 62.1% against L. brasiliensis, and from 66% to 78% against N. parvum, while the Trichoderma strains gave 61.2% to 81.1% inhibition of L. brasiliensis and close to 100% inhibition of N. parvum (Figure 3; Table 4).While T. harzianum T06BCMX did not affect radial colony growth of either of the pathogenic fungi, this isolate caused a significant decrease in aerial mycelium (Figure 3).

Characterization of mycoparasitic activity of Trichoderma strains
The pre-colonized plate experiments showed that all the assessed Trichoderma isolates had vigorous mycoparasitic activity, with colonization percentages ranging from 70% to 100% (Table 5).When the colonization percentage was 100%, the inoculated phytopathogenic fungus could not be recovered, indicating total suppression.Microscope observations from dual culture assays indicated that all the Trichoderma isolates coiled around, and cause morphological deformations, of L. brasiliensis hyphae, while the isolates T. asperellum T15BCMX and T. longibrachiatum T17BCMX also induced lysis of mycelium walls (Table 5; Figure 4).

Evaluation of biocontrol activity of selected bacterial and fungal isolates in greenhouse trials
The preventative application to soil of the Trichoderma and Bacillus isolates, for suppression of L. brasiliensis infection revealed the following.While the untreated   grapevine plants inoculated with L. brasiliensis developed wounds of mean length up to 10.0 cm, the plants treated with three isolates of T. asperellum (T20BCMX, EF09BCMX, and EF11BCMX), B. amyloliquefaciens BEVP26BCMX and Bacillus sp.rbES015 showed significantly shorter necrotic lesions (Figures 5 and 6).Plants inoculated with L. brasiliensis and treated with T. harzianum T6BCMX developed larger lesions than plants inoculated only with L. brasiliensis (Figures 5 and 6).This isolate was the only Trichoderma isolate that gave no effect in the diffusible compounds assays (Figure 3).In the greenhouse tests, all the Bacillus and Trichoderma isolates were recovered from the root tissues of the inoculated plants, and L. brasiliensis was re-isolated from the stems of the plants.

Evaluation of biocontrol activity of selected isolates under vineyard conditions
In the field assessments, grapevine branches preventively treated with most of the evaluated Bacillus and Trichoderma isolates showed two to five times shorter lesions than the untreated branches inoculated only with L. brasiliensis (Figure 7).Between the Trichoderma isolates no statistically significant differences were detected, while among Bacillus isolates, B. axarquiensis BEV-P02BCMX and B. amyloliquefacines BEVP26BCMX gave significant differences from B. amyloliquefacines BEV-P31BCMX.Isolate B. amyloliquefaciens BEVP26BCMX gave the strongest effect, with a 5-fold reduction on the length of the necrotic lesions (Figures 7 and 8).All the beneficial organisms were re-isolated from the respective treated branches, while L. brasiliensis was only re-isolated from the experimental controls and the Bacillus-treated, vines but not from the Trichoderma-treated branches.

DISCUSSION
This study has identified endophytic microorganisms associated with heritage grapevines that biocontrol L. brasiliensis and N. parvum, which are two of the most virulent fungi associated with Botryosphaeria dieback (Gramaje et al., 2018;Rangel-Montoya et al., 2021).The isolates used in this research were originally obtained from GTD symptomatic grapevines growing in the Guadalupe Valley in Baja California, Mexico (Rangel- Montoya et al., 2021).Considering the conditions in which heritage vines grow in this region, with no irrigation and with little cultural management, the study has identified beneficial microorganisms that could be useful in commercial vineyards.This is the first study focusing on biological control potential of microorganisms associated with heritage grapevines.
Under in vitro conditions, several Bacillus isolates obtained from heritage grapevines inhibited L. brasilien- sis and N. parvum, and showed additional characteristics associated with plant growth promotion, including production of siderophores and indole acetic acid, and solubilization of phosphate and potassium.In dual culture assays five Bacillus strains inhibited growth of L. brasiliensis by up to 51%, showing that these assays were useful for the initial screening and selection of strains with antagonistic activity.This method was previously shown to be useful for identifying two Bacillus strains with antagonistic effects against Diaporthe ampelina, Diplodia seriata, Eutypa lata and N. parvum (Blundell et al., 2021), and two strains of Bacillus velezensis against eight different fungi, including L. theobromae, D. seriata and N. parvum (Bustamante et al., 2022).
While Bacillus spp.inhibited the growth of N. parvum by up to 34% through the production of organic volatile compounds, L. brasiliensis was not affected.However, a decrease in formation of aerial mycelium was observed, indicating a slight antagonistic effect on this fungus.However, diffusible compounds had strong antifungal effects against both pathogenic fungi, although these were less against L. brasiliensis (from 40% to 62%), indicating that diffusible compounds were the main antagonistic mechanisms of these Bacillus isolates.The production of volatile antifungal compounds, such as ketones, alcohols, esters, pyrazine, acids, hydrocarbons, heterocycles, aldehydes, phenols, thioalcohols, and thioesters, has been reported for Bacillus spp.For example, B. amyloliquefaciens CPA-8 produced 1,3 pentadiene, acetoin (3-hydroxy-2 butanone), and thiophene, that reduced in vitro mycelial growth of Monilina laxa, M. fructicola, and Botrytis cinerea (Gotor-Vila et al., 2017).Diffusible compounds with antifungal activity have also been identified from Bacillus spp., including iturin, fengycin, macrolactin, surfactin.Bacillus INECOL-6004, INECOL-6005, and INECOL-6006 showed antagonistic activity against Fusarium kuroshium by the production of iturin, fengycin, and surfactin (Guevara-Avedaño et al., 2020).Identifying the metabolites produced by the Bacillus isolates obtained in the present study, and their role in disease suppression, would be worthwhile.
All of the seven selected Trichoderma strains showed in vitro antagonistic activity.Additionally, they all produced siderophores, chitinase, and indole acetic acid.Several previous studies have identified Trichoderma spp. with activity against GTD pathogens.Úrbez-Torres et al., (2020) evaluated the antagonistic activities of sixteen Trichoderma strains against D. seriata, E. lata and N. parvum, with T. atroviride PARC1018 giving the greatest inhibition of D. seriata and E. lata, and T. koningiopsis against N. parvum.Similarly, Blundell et al., (2021) reported that T. asperellum UC8360 inhibited D. seriata, E. lata, N. parvum and D. ampelina, with D. ampelina being the most inhibitory, while a T. harzianum isolate gave high rates of inhibition of N. parvum (Langa-Lomba et al. 2022).In the present study, three strains showed inhibition proportions greater that 50%.Two of these (T.asperellum EF09BCMX and T. asperellum EF011BCMX) were isolated from heritage grapevines, indicating that the heritage grapevine cv.'Mission' contains beneficial microorganisms that can be used as BCAs with additional benefit of plant growth promotion.Different Trichoderma strains inhibit growth of phytopathogenic fungi through the production of volatile compounds (Zhao et al., 2022), In the present study, however, the selected strains did not inhibit the mycelial growth of L. brasiliensis or N. parvum by the production of volatile compounds, although decreases in the formation of aerial mycelium were observed.
Mycoparasitism is considered to be an important biocontrol mechanism of Trichoderma (Sood et al., 2020).However, previous studies showed that this mode of action is not always present.For example, from 50 Trichoderma isolates evaluated against Moniliophthora roreri, mycoparasitism varied between 0% and 100%, with only nine isolates reaching 100% (Reyes-Figueroa et al., 2016).Leiva et al. (2020) also found that for 199 Trichoderma isolates, mycoparasitism rates varied from 32% to 100%.Isolates with this capacity parasitize and colonize phytopathogens, reducing the fungal inoculum and alleviating the intensity of the diseases they cause (Nusaibah andMusa, 2019, Mukherjee al., 2022).The pre-colonized plate assays of the present study showed a rates of L. brasiliensis from 70% to 100%.When T. asperellum EF09BCMX and T. asperellum T15BCMX were evaluated, L. brasiliensis was not recovered from the plates, indicating total elimination of the pathogen, as expected by the 100% colonization obtained.In contrast, the microscopical observations showed that hyphae from seven Trichoderma isolates coiled around hyphae of L. brasiliensis.This ability has been extensively reported.For example, T. asperellum UDEAGIEM-H01 formed coils around hyphae of F. oxysporum and Macrophomina phaseolina (Díaz-Gutiérrez et al., 2021), T. harzianum KMISO2-2-19A around Fusarium virguliforme hyphae (Pimentel et al., 2020), and T. koningiopsis around hyphe of Phytophthora xcambivora (Frascella et al., 2022).Coiling around hyphae is the first step of Trichoderma mycoparasitic activity, and is followed by production of hydrolytic enzymes that allow Trichoderma to penetrate the hosts and absorb their contents (Rocha-Ramirez et al., 2002).Trichoderma isolates produce extracellular cell wall degrading enzymes such as endochitinases, b-1,3-glucanases, and proteases, that lyse pathogen mycelium (Harman et al., 2004;Druzhinina et al., 2011).Although the production of cell wall degrading enzymes was not evaluated in the present study, T. asperellum T15BCMX and T. longibrachiatum T17BCMX caused deformation and the cell wall lysis of L. brasiliensis hyphae, indicating production of enzymes that damaged cell walls, and potential as BCAs.
Although the in vitro tests provided information on the antagonistic potential of the evaluated strains, low inhibition proportions may not indicate that isolates will perform poorly when applied as biocontrol agents under field conditions.Effectiveness of biocontrol in the field often depends on capacity to colonize plant tissues, establish compatible interactions, prevail in the hosts, and tolerate abiotic factors (Finkel et al., 2017;Afzal et al., 2019).For example, isolate B. axarquiensis BEVP02BCMX showed low inhibition in dual culture assays against L. brasiliensis, but when it was applied as a preventive pruning protectant, it reduced the size of the necrotic lesions.Effectiveness of this strain could be related to its ability to colonize grapevine tissues, which is possibly expected considering its endophytic nature.However, beneficial microorganisms do not always reduce damage caused by pathogens, as was observed here with T. harzianum T06BCMX, since plants showed longer necrotic lesions compared to those inoculated only with L. brasiliensis.This effect has been observed previously.Leal et al. (2021) reported that plants inoculated with T. atroviride SC1 and B. subtilis PTA-271, applied in soil against N. parvum, developed longer necrotic lesions than experimental controls.This is a good reason why potential biological control agents should be thoroughly assessed.
In plants, beneficial microorganisms have been evaluated using different methods, including preventative applications in substrates or directly applied to pruning wounds (Haidar et al., 2016).In the present study, selected isolates were first evaluated by direct application to soil, and then in a vineyard by applying them to pruning wounds.In the greenhouse assay, two Bacillus isolates (Bacillus sp.rbES015 and B. amyloliquefaciens BEVP26BCMX) and three T. asperellum isolates (EF09B-CMX, EF11BCMX, and T20BCMX) reduced necrotic lesion lengths caused by L. brasiliensis.Since the beneficial microorganisms were applied in soil without direct contact with the pathogen, the observed effect could be due to activation of host systemic response.Previous studies have indicated that non-pathogenic bacteria and fungi have capabilities to reduce damage caused by pathogens through activation of induced host systemic resistance.Haidar et al. (2016) and Zehra et al. (2021) identified different bacteria isolates with biocontrol activity against P. chlamydospora, when applied preventatively as drenches.Similarly, Haidar et al. (2021) identified different bacteria isolates that reduced necrotic lesions caused by N. parvum in grapevines when inoculated in soil.This opens the possibility for applying selected biocontrol isolates during irrigation or as drenches, diminishing the costs of biocontrol applications.
Pathogens causing GTDs enter grapevines mainly through pruning wounds (Gramaje et al., 2018), so control strategies should focus on wound protection.Few studies have been carried out in field conditions, and they generally used commercial formulations and specific strains.Martínez-Diz et al. (2020) evaluated the Trichoderma isolates SC1 and 1-1237 against D. seriata and P. chlamydospora, observing low efficacy.The low effectiveness of commercial formulations based on biocontrol agents is common, and has been mainly attributed to microorganism failure to colonize plant tissues (Mutawila et al., 2016).In the present study, eight isolates applied directly to grapevine wounds reduced necrotic lesions caused by L. brasiliensis, and only Bacillus sp.rbES015 failed.This isolate was obtained from soil so may be incapable of colonizing grapevine tissues.The microorganisms obtained in the present study showed strong biocontrol activity, even though they applied without the addition of carriers and protectants.Although comprehensive evaluation needs to be carried out, it may be possible to transfer the selected strains to a company or association to develop a formulation based on the strains to improve its usability and stability for grape producers.
In this research, a strain of T. longibrachiatum was shown to be a good candidate as a biological control agent.However, this species has been reported as an opportunistic human pathogen of immunosuppressed patients (Myoken et al., 2002;Lipový et al., 2021;Vasiliki et al., 2021).Therefore, the use of T. longibrachiatum T17BCMX as biological control agent should be restricted, though it showed excellent antagonistic activity in assays carried out under in vitro and in planta conditions.Most of the other identified Trichoderma strains were T. asperellum, which is widely used as a biological control agent, without reported human risks.
The antagonistic activity of the Bacillus isolates was mainly due to production of diffusible compounds, while in the Trichoderma spp. it resulted from production of diffusible compounds and the mycoparasitism.However, volatile compounds may also have contributed to the biological control activity of the isolates, since all the selected strains produced these compounds that inhibited Neofusicoccum in vitro.
In the present study strains with antagonistic activity were tested separately.Combination of isolates of Bacillus and Trichoderma have been previously shown to be successful.For example, B. subtilis PTA-271 and T. atroviride SC1 were evaluated individually and together against N. parvum Bt67 in two grapevine varieties ('Tempranillo' and 'Chardonnay').'Tempranillo' plants inoculated with either T. atroviride SC1 or the consortium had fewer internal lesions caused by N. parvum (Leal et al., 2021).In the future, compatibility among the isolates identified in the present study could be determined for the development of a consortium that takes advantage of the strengths of different isolates.
In conclusion, this study has identified Bacillus and Trichoderma isolates with biocontrol activity against L. brasiliensis when applied preventatively to soil and to the pruning wounds.Therefore, heritage grapevines of Baja California have been shown to be a reservoir of beneficial microorganisms, which can be potentially utilized in commercial grapevine varieties to help reduce damage caused by grapevine trunk disease fungi.

Figure 1 .
Figure 1.Representative images of dual culture assays of selected Bacillus and Trichoderma isolates against Lasiodiplodia brasiliensis.

Figure 2 .
Figure 2. Representative images of the antifungal effects of volatile organic compounds produced by Bacillus and Trichoderma isolates against Lasiodiplodia brasiliensis and Neofusioccum parvum.

Figure 3 .
Figure 3. Representative images of the antifungal effect of diffusible organic compounds produced by Bacillus and Trichoderma isolates against Lasiodiplodia brasiliensis and Neofusicoccum parvum.

Figure 4 .
Figure 4. Representative microscope images (captured after 28 d incubation at 30ºC) from pre-colonization assays of Trichoderma isolates against Lasiodiplodia brasiliensis showing mycoparasitism activity of the Trichoderma isolates (red arrows indicate effects caused by Trichoderma).A, Hyphal coiling of T. asperellum EF11BCMX against L. brasiliensis.B, T. harzianum T06BCMX causing deformation in L. brasiliensis hyphae.C, Lysis of the hyphal wall of L. brasiliensis induced by T. longibrachiatum T17BCMX.

Figure 6 .
Figure 6.Images of grapevine stems after preventive soil inoculations with Bacillus or Trichoderma isolates and Lasiodiplodia brasiliensis.The yellow lines indicate the lengths of necrotic lesions caused by L. brasiliensis in 'Cabernet Sauvignon' stems.

Figure 8 .
Figure 8. Images field-grown grapevine stems after treatments of pruning wounds with different Bacillus or Trichoderma isolates against Lasiodiplodia brasiliensis infections.The images are of ten replicates representative plants, taken of 30 d after L. brasiliensis inoculations.

Table 1 .
GenBank and culture accession numbers of bacterium species used in the present study for phylogenetic analyses.

Table 2 .
List of GenBank and culture accession numbers of fungal species used in the present study for phylogenetic analyses.

Table 3 .
Mean percent inhibition of Lasiodiplodia brasiliensis by different potential biocontrol microorganisms, and their respective production of plant growth promotion compounds, for selected Bacillus and Trichoderma isolates.

Table 4 .
Mean percent inhibition of Lasiodiplodia brasiliensis and Neofusicoccum parvum from volatile organic compounds and diffusible organic compounds by different Bacillus and Trichoderma isolates.

Table 5 .
Mean colonization percentages from pre-colonized plate assays, and microscope observations, indicating mycoparasitism activity of six Trichoderma isolates against Lasiodiplodia brasiliensis.