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Article

Use of Bacteria Isolated from Berry Rhizospheres as Biocontrol Agents for Charcoal Rot and Root-Knot Nematode Strawberry Diseases

by
María Camacho
1,*,
Berta de los Santos
1,
María Dolores Vela
2 and
Miguel Talavera
3
1
Andalusian Institute of Agricultural and Fisheries Research and Training (IFAPA), IFAPA Centro Las Torres, Carretera Sevilla-Cazalla Km. 12.2, 41200 Alcalá del Río, Sevilla, Spain
2
Andalusian Institute of Agricultural and Fisheries Research and Training (IFAPA), IFAPA Centro Rancho de la Merced, Carretera Cañada de la Loba Km. 3,1, 11471 Jerez de la Frontera, Cádiz, Spain
3
Andalusian Institute of Agricultural and Fisheries Research and Training (IFAPA), IFAPA Centro Alameda del Obispo, Av. Menéndez Pidal s/n, 14004 Córdoba, Córdoba, Spain
*
Author to whom correspondence should be addressed.
Horticulturae 2023, 9(3), 346; https://doi.org/10.3390/horticulturae9030346
Submission received: 7 February 2023 / Revised: 1 March 2023 / Accepted: 3 March 2023 / Published: 6 March 2023
(This article belongs to the Special Issue Plant-Parasitic Nematodes in Horticultural Crops)
Browse Figures
Versions Notes

Abstract

:
Strawberry is a high-value crop in Spain, where fruits for fresh consumption are produced off-season and exported to central and northern European countries. Soil-borne pathogens (SBP), such as Macrophomina phaseolina and Meloidogyne hapla, are widely spread in strawberry crops in Spain. The prevalence of these pathogens is a significant barrier to producing strawberries of premium quality and at profitable yields. The current ban on fumigant pesticides drives the search for alternative SBP control methods that can simultaneously control both SBP. Twenty-nine bacterial strains were isolated and identified from strawberry and blueberry rhizospheres and screened for plant growth promotion activities as well as for biocontrol potential on M. phaseolina and M. hapla. Two of these bacterial strains (Bacillus velezensis FC37 and Pseudomonas aeruginosa AC17), together with Brevibacterium frigoritolerans Hvs8 coming from IFAPA’s bacterial collection, were selected and evaluated in planta for their biocontrol potential on strawberry SBP diseases caused by M. phaseolina and M. hapla. The three tested bacterial strains reduced charcoal rot disease severity, crown incidence and severity, and petiole colonization by M. phaseolina. Root-knot nematode (RKN) symptoms were reduced by P. aeruginosa AC17 and B. velezensis FC37, but RKN reproduction was only reduced in plants inoculated with P. aeruginosa AC17. Pseudomonas aeruginosa AC17 showed the greatest potential as a biocontrol agent candidate to be included in integrated disease management programs to control the two most prevalent soil-borne pathogens of strawberry in Spain.

1. Introduction

Strawberry is a high-value crop in Spain, where fruits for fresh consumption are produced off-season and exported to northern European countries. Up to 93% of all strawberries produced in Spain are grown in the southwestern region (Huelva province), where 6867 ha produce 377,596 tons with a market value of EUR 392 million [1]. Spain produces between 25 and 33 percent of all strawberries grown in Europe. In intensive monoculture systems, strawberries are produced in yearly crop cycles from October to May under temporary plastic high tunnels. The fields are left fallow from July to September, while the harvest season runs from January to late May. Because of the extended strawberry cropping cycles and high farm productivity and profitability, crop rotation with other crops is not practical.
Due to monoculture, soil-borne pathogens (SBP), such as fungi Macrophomina phaseolina and Fusarium spp. [2,3,4,5], as well as nematodes, such as Meloidogyne hapla, Pratylenchus penetrans, and Hemicycliophora spp. [6], are widespread in strawberry crops in Spain, with prevalence ranging from 60 to 80%. Strawberry yield losses due to SBP have been estimated at 10% in case of fungi and 6% for nematodes. As such, they are a significant barrier to producing strawberries of top quality with commercially sustainable yields [7]. There is no resistance reported in strawberry against these pathogens, and soil disinfestation with fumigant agrochemicals (1,3-dichloropropene, chloropicrin, metam-Na, or metam-K) has been the main strategy for SBP control in the area [8]. The continued use of soil chemical fumigants has a negative impact on soil biodiversity, accumulates residue with a significant risk of contaminating groundwater, and causes environmental issues. Therefore, the majority of soil fumigants are currently illegal or subject to tight regulations both inside and outside of the European Union (Directive 2009/128/CE). For the management of soil-borne diseases in vegetables, extensive research has been performed on different chemical and non-chemical SBP control strategies, such as solarization, biofumigation, biosolarization, and steaming. Nevertheless, none of these techniques are as effective as soil fumigation at preventing SBP, and they have not been shown to be reliable enough to be utilized in intensive horticulture production. [9]. As result, under the current regulatory framework, and with the exception of temporary authorizations, farmers do not have a soil disinfestation option effective enough to reduce high soil infestations by SBP, which seriously affect the productivity of these intensive strawberry crops. Thus, the search for environmentally friendly alternatives, among which the use of microorganisms stands out, is increasingly crucial.
The use of autochthonous biocontrol organisms (BCO) as a strategy for controlling pests and diseases is an established technique for controlling pests but less frequently used in controlling SBP. Given the need to avoid the introduction of allochthonous microorganisms that could become an ecological problem as an invasive species, biopesticides should be based on microbial strains identified locally. After isolation, some microorganisms are selected for their plant growth-promoting properties or for their biocontrol potential in vitro. They can function as homologous of the pathogen, release substances against them, or even induce a resistance response to the pathogen in the plant, helping in combination with other control methods within an integrated disease management strategy. The best biocontrol candidates are also characterized by 16sRNA sequencing to know the species (or genera) at which they are assigned in order to avoid species that could be of any risk to humans, animals, and/or plants.
Several beneficial bacteria have been shown to be effective against SBP, although the most frequently reported strains come from the genera Bacillus and Pseudomonas [10]. Their bioactive secondary metabolites act through several mechanisms, including plant growth promotion, induction of systemic resistance, changes in root exudates, and production of phytohormones, antibiotic substances, and volatile organic compounds [11]. The most frequently explored rhizobacterial metabolites with inhibitory activity against various SBP are extracellular enzymes, such as proteases and chitinases. The lipid layer required for the growth and preservation of nematode eggs was discovered to be destroyed by those compounds acting together, which had an effect on both nematode development and hatching, suggesting that they could act as biocontrol agents for soil pathogenic fungi and phytoparasitic nematodes at the same time [10].
Furthermore, some Bacillus and Pseudomonas strains are good BCO candidates because of their easy production, transport, and application [12], and because they have been recently proposed as good candidates for BCO for Meloidogyne [13,14] and M. phaseolina [15]. Most reports indicate biocontrol potential against a single SBP but reports of bacterial strains active in biocontrol of two or more SBP are scarce. Because many of the biocontrol mechanisms of these BCO are active against different SBP, screening for BCO active simultaneously against several SBP present in a region is of particular interest.
The purpose of this study was to identify possible BCO from berry rhizospheres in the southwest Spain strawberry growing region and to test their efficacy as biocontrol agents against M. hapla and M. phaseolina under in vitro and in planta conditions.

2. Materials and Methods

2.1. In Vitro Experiments for Biocontrol of M. phaseolina

2.1.1. Bacterial Strain Isolation

Bacterial strains occurring in strawberry and blueberry rhizospheres at the IFAPA experimental station “El Cebollar” (Moguer, Huelva, Spain) (37°14′ N–06°48′ W) were isolated and maintained at the IFAPA Las Torres bacterial collection. For strain isolation, serial ten-fold dilutions of 1 g of rhizosphere soil homogenized in 99 mL of mineral salt were achieved. The dilutions were spread on Plate Count Agar (PCA, Difco®, Madrid, Spain) supplemented with cycloheximide (100 mg/L, Sigma-Aldrich®, St. Louis, MO, USA) and incubated at 28 °C for 24–48 h until the appearance of individual colonies. For obtaining pure strains, colonies with different morphotypes were further streaked on individual plates. Strains were further cryo-preserved at −80 °C in 15% glycerol-0.5% peptone.

2.1.2. Bacterial Strain Identification

Bacillus velezensis FC37 and Brevibacterium frigoritolerans Hvs8 had been previously identified and characterized [16]. For identification of the remaining strains, the 16S rRNA coding gene was amplified by PCR using universal primers 27F (5’-AGA GTT TGA TCC TGG CTC AG-3’) and 1492R (5’-GGT TAC CTT GTTACG ACT T-3’) [17]. Amplification was carried out in a thermicycler MyiQ™ (Bio-rad, Dubai, United Arab Emirates) as follows: denaturation at 95 °C for 5 min followed by 30 s at 94 °C, 30 s at 50 °C, 1 min at 52 °C, and 1 min at 72 °C, with a final extension at 72 °C for 7 min at the end of 35 cycles. Amplification and sequencing of purified amplicons was conducted by Universidad de Leon (Spain). To compare similarities with known sequences, the EzBioCloud database (https://www.ezbiocloud.net accessed on 16 January 2023) was used [18]. The 16S rRNA gene sequences were deposited in the DDBK/EMBL/GenBank database under the accession numbers OQ255851- OQ255878 (strains AC11.2, AC14, AC15, AC17, AC9.1, ACH10, ACH14, ACH15, ACH16, ACH2, ACH25, ACH29, FC10.1, FC10.2, FC14, FC15, FC17, FC18, FC20, FC21.2, FC25, FC28, FC32, FC34, FC35, FC36, FC39, FC5.2, respectively).

2.1.3. In Vitro Assays for Plant Growth-Promoting Activities

Strains were evaluated in vitro for plant growth-promoting abilities. Indolacetic (IAA) production was assessed in TSB (tryptic soy broth) supplemented with L-tryptophan (100 mg/L) and was measured in the supernatant after incubation at 28 °C with continuous shaking for 72 h [19]. Siderophore production was assessed in CAS plates following the protocol of Alexander and Zuberer [20], and phosphate solubilization was determinate in PVK plates following the procedure described by de Freitas et al. [21]. All assays were repeated twice. Activities were assessed visually from no activity (-) to very strong activity (++++). For graphical representation, a strain showing very strong activity (++++) in all the three measured properties will reach 100% (33% for each maximum activity). The other data were normalised accordingly.

2.1.4. In Vitro Assays for Biocontrol Enzymatic Activities

Strains were evaluated in vitro for the biocontrol enzymatic activities of cellulase, protease, amylase, β-glucosidase, and chitinase, as described by Viejobueno et al. [15]. The presence of volatile compounds (HCN) was estimated qualitatively by the sulfocyanate colorimetric method [15]. All assays were repeated twice. Graphical representation was performed as described before.

2.1.5. In Vitro Assays for Biocontrol Activity against M. phaseolina

In vitro assays were performed in 6 cm diameter Petri plates containing TSA medium, which is TSB medium plus 16 g/L of bacteriological agar (Difco®, Madrid, Spain). Four droplets of inoculum (10 µL) of each bacterial strain were distributed at the four perpendicular directions at the periphery of the plate and incubated at 28 °C for 24 h. Then, mycelium discs of M. phaseolina isolates TOR-102 or TOR-872 were placed in the center of the plate and incubated for 7 more days at 28 °C. Plates without bacteria inoculum were used as negative controls. Percentage of inhibition of pathogen development compared to the control was assessed at seven days after confrontation and was calculated using Formula (1):
Percentage of inhibition (%) = [(Rc − Ri)/Rc] × 100
where Rc is the radial growth of the fungal pathogen in the control plates (mm) and Ri is the radial growth of the fungal pathogen in the test plates (mm). Experiments were conducted in triplicates.

2.2. In Planta Experiments for Biocontrol

The effects of both bacterial inoculation on the development of strawberry (Fragaria × ananassa Duch., ‘Rociera’) and the control of strawberry soil-borne diseases were evaluated in pot tests conducted in a growing chamber. Three antagonist biocontrol bacteria (BCO) were selected for their biocontrol potential in vitro against M. phaseolina and their plant growth-promoting properties. Bacillus velezensis FC37, Brevibacterium frigoritolerans Hvs8, and Pseudomonas aeruginosa AC17 were tested in planta against the two most prevalent SBP in the strawberry growing area of southwestern Spain: M. phaseolina and M. hapla.

2.2.1. Experimental Design

Twelve treatments were used in each pot test, divided into two components with three and four levels, respectively: SBP (M. hapla/M. phaseolina/non-SBP) and BCO (B. velezensis/B. frigoritolerans/P. aeruginosa/non-BCO) (Table 1). In the growing chamber, pots were placed in a completely randomized design, with each treatment being replicated eight times. Two experiments were conducted.

2.2.2. Plant Material

Strawberry (‘Rociera’) transplants were obtained from high-altitude nurseries and stored at 4 °C until the establishment of the pot trials. Twenty-four hours before planting, the roots of all plants were cut and immersed in 3.5% sodium hypochlorite solution for 5 min, washed under running tap water, dried in absorbent paper, and put back into storage at 4 °C until planting in the pots.

2.2.3. Bacterial Inoculation

A final concentration of 109 CFU/mL was achieved by growing the selected bacterial strains in tubes containing 5 mL of Trypto-casein Soy Broth (TSB) (Scharlap®, Barcelona, Spain) that were incubated at 28 °C for 72 h while being continuously stirred (180 rpm). For inoculation on plants, cultures were centrifuged at 12,000 rpm for 5 min and the cells were resuspended in sterile 0.03 M magnesium sulfate buffer to a concentration of 108 CFU/mL.
Strawberry seedlings were inoculated at planting by root immersion in a solution of each bacteria strain (108 bacteria/mL for 1 h). Under the same conditions, control plants were submerged in sterile 0.03 M magnesium sulfate buffer. Seven days after planting, a reminder dose with bacteria (10 mL of a 108 bacteria/mL solution) was applied by irrigation onto the soil surface of each pot.

2.2.4. Fungal Inoculation

Eight days after planting, strawberry plants were infected with M. phaseolina isolate TOR-102 from pure cultures from the IFAPA’s fungal collection. To recover the virulence of M. phaseolina TOR-102, it was grown on soybean (Glycine max ‘Osumi 2004’) seeds from the collection of the IFAPA Center Las Torres [22]. Each pot received 50 mL of a suspension containing 2 × 103 sclerotia of M. phaseolina TOR-102 per milliliter [23,24].

2.2.5. Nematode Inoculation

A population of Meloidogyne hapla isolated from infected strawberry roots was used for the growing chamber tests. To promote nematode proliferation, tomato plants of the ‘Roma’ variety (Solanum lycopersicum) were infected with the nematode and kept for 8 weeks in a growth chamber at 25 °C. The diseased tomato roots were stirred in a 0.5% sodium hypochlorite solution to remove nematode eggs [25]. To obtain second-stage juveniles (J2), the egg suspension was concentrated on a 20 µm filter and deposited on Baermann funnels [26]. Only juveniles hatched within 24–48 h were used as inoculum, whereas J2 hatchings within the first 24 h were eliminated. Nematode inoculation was carried out eight days after planting by pouring an aqueous suspension of 1500 J2 of M. hapla per pot through three 5 cm deep holes around the strawberry plant.

2.2.6. Plant Growing Conditions

Strawberry plants were grown singly in polypropylene pots that were 11 cm tall, 12 cm in diameter at the top, and 10 cm in diameter at the bottom. The pots also contained 750 mL of peat substrate (Indalofertil premium ®), which had been previously sterilized in an autoclave twice on consecutive days at 120 °C and 1 atmosphere for 30 min. The plants were placed in the growing chamber at random and allowed to grow with an average daytime temperature of 28 °C, a nighttime temperature of 22 °C, and a photoperiod of 16 h of light. All pots were fertilized by adding 2 g of Osmocote® (15% N + 10% P2O5 + 12% K2O + 2% MgO2 + microelement, Scotts Company, Heerlen, the Netherlands) on the soil surface. The pots were watered according to requirements with 50–100 mL of water once or twice a week, maintaining a slight water stress. Plants were grown for 98 days in 2019–2020 and 159 days in 2020–2021 experiments.

2.2.7. Parameters Evaluation for Plant Growth and Disease

Weekly observations were employed to follow the progression of charcoal rot caused by M. phaseolina. The incidence of charcoal rot was calculated as the proportion of dead plants to the total. Disease severity was estimated using a scale from 0: healthy plant to 4: dead plant [27]. To confirm infection by the pathogen, isolations from symptomatic tissues were performed on culture medium potato dextrose agar, keeping the Petri dishes at 30 °C and in darkness for 7 days. The percentage of M. phaseolina-infected plants (roots or petioles), crown incidence, and severity of symptoms (scale 0 to 5) were estimated [28].
At harvest, strawberry plants were uprooted, washed to remove soil debris, and their fresh and dry weights were determined, including stolons, fruits, and roots.
At the end of each test, root-knot nematode disease was evaluated by assessing the severity of the nodulation symptoms in the roots using a gall index on a 0–10 grading scale (0 = no galls; 7 = 100% of the roots had galls; 10 = dead plant) [29], the final populations of nematodes per pot, and different growth parameters of the plant. Three grams of root subsamples were cut into pieces 1–2 cm long and macerated by blending in a 1% solution of sodium hypochlorite to determine the nematode population in the roots [25]. For gathering M. hapla eggs, juveniles, and adults, the suspension was poured into sieves with a 20-µm mesh. Nematodes were counted in counting plates and the total number of nematodes in the entire root system was obtained by multiplying the nematodes count per g of roots by the root fresh weight. Nematodes were isolated from 250 cm3 of soil using the sieve and decanting method and then centrifuged in a solution of magnesium sulfate heptahydrate with a specific gravity of 1.18 g/L to determine the nematode population density in the soil [30]. For further counting under a microscope, the nematode suspension was concentrated in 2 mL of water. Final nematode population densities were calculated as the sum of nematodes extracted from roots plus those extracted from soil (750 mL) and were expressed as nematodes per pot.

2.3. Statistics

For in vitro growth inhibition of fungal strains, a Kruskal–Wallis test was carried out using the program Statistix 9.1 to assess the inhibition of the bacterial strains on the growth of M. phaseolina. Pairwise comparisons were performed using an alpha of 0.05.
All results concerning plant tests are expressed as mean ± standard error of the mean. The statistical program Statgraphics Centurion XVI® (Statpoint Technologies Inc., Warrenton, VA, USA) was used to analyze the data. Data were subjected to the Kolmogorov–Smirnov and Brown–Forsythe tests to determine whether the variances were normal and homoscedastic; if significant, data were arcsine-transformed and tested again. Data were analyzed using ANOVA when normality and homoscedasticity of variances could be assumed. The HSD Tukey’s test (p < 0.05) was used to compare the means if the F values were significant. When the homoscedasticity of variances could not be assumed, Welch’s ANOVA was used. The data were analyzed using Kruskal–Wallis non-parametric tests when normality was not achieved after transformation. If H values were significant, means were compared by Dunn’s multiple comparison test (p< 0.05).

3. Results

3.1. Bacterial Identification

Twenty-nine bacterial strains were isolated from strawberry (FC codes) and blueberry (AC codes) rhizospheres from IFAPA El Cebollar berry crops and taxonomically identified using their 16S rRNA gene sequences (Table 2).

3.2. Plant Growth-Promoting Activities

Plant growth-promoting abilities in selected bacterial strains as synthesis of auxins, siderophores, and phosphate solubilization are shown in Figure 1. Two strains isolated from strawberry rhizosphere (Pseudomonas kribbensis FC5.2 and Bacillus velezensis FC37) and five from blueberry (Mixta calida AC14, Enterobacter quasiroggenkampii AC15, Chryseobacterium cucumelis ACH2, Klebsiella variicola ACH10, and Enterobacter kobei ACH25) displayed the three tested properties. Klebsiella variicola ACH10 showed the best results. It is noticeable that strains from blueberry rhizosphere showed more plant growth-promoting activities and with higher values than those from strawberry rhizosphere.

3.3. Biocontrol Enzymatic Activities

The biocontrol-related enzymatic activities (β-glucosidase, amylase, cellulase, chitinase, and casein) measured in the selected bacterial strains are shown in Figure 2. None of the isolated strains displayed all biocontrol activities or HCN production. The strain Bacillus albus FC32 was the only one that displayed chitinase activity. This strain, together with strains Xanthomonas translucens FC10.1, B. velezensis FC37, Microbacterium arborescens ACH15, and B. velezensis ACH16, showed four out of five biocontrol activities. Some strains from strawberry rhizospheres did not show any biocontrol activities.

3.4. In Vitro Antifungal Activities against M. phaseolina

M. phaseolina growth inhibition was assessed on TOR-102 and TOR 872 isolates. Most strains were able to inhibit M. phaseolina growth at some level, but B. velezensis FC37, P. aeruginosa AC17, and B. velezensis ACH16 totally inhibited the growth of both fungi isolates (Figure 3). We found a significant effect of the bacterial strains on the inhibition of M. phaseolina (H (28) = 118.5, p < 0.001).

3.5. Strawberry Biomass

Plant biomass was reduced by 29.3% in strawberry plants infected with M. phaseolina (38.9 ± 2.2 g) when compared to non-SBP-inoculated plants (55.0 ± 2.8 g) (p < 0.05). Strawberry infection with M. hapla (52.0 ± 5.4 g) and bacterial inoculation with B. frigoritolerans Hvs8 (46.4 ± 3.6 g), P. aeruginosa AC17 (46.6 ± 2.3 g), and B. velezensis FC37 (43.3 ± 2.8 g) did not have any effect on plant biomass (p > 0.05).

3.6. M. phaseolina Disease Symptoms

Macrophomina phaseolina caused the death of 12.5% of the plants in the pot experiments when no BCO was inoculated, but no dead plants were found in M. phaseolina-infected strawberry plants if they were inoculated with P. aeruginosa AC17. The three tested bacterial strains reduced plant disease severity, crown incidence and severity, and petiole colonization by M. phaseolina. Root colonization by M. phaseolina was not affected by any BCO inoculation (Table 3).

3.7. M. hapla Disease Symptoms

Root-knot nematode symptom severity was reduced by P. aeruginosa AC17 and B. velezensis FC37, but RKN reproduction was only reduced in plants inoculated with P. aeruginosa AC17 (Table 4).

4. Discussion

Selection of biocontrol agents should include an isolation process from the ecological niche where they will be used, screening for their plant promotion interactions and biocontrol properties under in vitro conditions, and, finally, in vivo testing to assess the desirable properties for which they have been chosen. Furthermore, it is desirable that the selected strains can grow quickly and in a high number in synthetic media for an easy formulation. In this study, strains have been isolated from the rhizosphere of healthy blueberry and strawberry plants growing at Moguer (Huelva, Spain). All isolates have been characterized by their in vitro properties, and those able to control the growth of the pathogens under in vitro conditions have been chosen for further in vivo testing. All selected strains belong to species frequently used in bioinoculant formulations (Bacillus and Pseudomonas) for their easy growth and formulation capacity [31,32]. Intriguingly, strains isolated from blueberry rhizosphere displayed more plant growth-promoting properties and with higher values than those from strawberry. Similarly, some of the strains from strawberry rhizosphere did not show biocontrol properties at all. Blueberry is a perennial crop, and we chose healthy plants from an area with a high incidence of M. phaseolina for bacterial isolation. It could be that these rhizospheric bacteria were responsible for the better growth displayed by these plants.
Biocontrol of M. phaseolina by autochthonous bacteria has been reported for several crops. The use of biopesticides based on native Bacillus strains has proven effective in controlling charcoal rot in geranium [33] and strawberry [34,35]. Inoculation with Azospirillum brasilense REC3 strain induced a defense response in strawberry plants against virulent isolates of M. phaseolina and evidenced an increased tolerance to charcoal rot disease [36,37]. In the same studies, native Bacillus and Pseudomonas spp. have been reported as biocontrol agents of root-knot nematodes in several crops [13,38,39,40]. Although it is desirable to use autochthonous bacteria, use of bacteria isolated from similar and nearby ecosystems for biocontrol purposes has also been reported. Thus, some genera, mainly Pseudomonas and Bacillus, have showed biocontrol activities against strawberry charcoal rot in southern Spain [15]
We report reductions in disease symptoms for both strawberry pathogens (M. phaseolina and M. hapla) by the P. aeruginosa AC17 strain. Strawberry petiole colonization by M. phaseolina and M. hapla reproduction was reduced in P. aeruginosa AC17-inoculated plants. Pseudomonas aeruginosa is a beneficial soil bacterium that promotes plant growth and development through production of a variety of regulatory compounds (IAA, ammonia, polysaccharides, and phosphate solubilization) directly in the rhizosphere and acts as a BCO directly by altering plant hormone levels or reducing the potential of SBP. P. aeruginosa also exhibited strong antagonistic properties against M. phaseolina in other crops, such as chir pine [41]. The most common molecules involved in this mechanism are, among others, siderophore, IAA, phenazines, sessilins, orfamides, chitinases, glucanases, and proteases [10]. Pseudomonas aeruginosa AC17 produces siderophores and proteases, while other isolated strains able to totally inhibit in vitro growth of M phaseolina (B. velezensis FC37 and B. velezensis ACH16) displayed much greater biocontrol capacities. It could be that P. aeruginosa AC17 produced secondary metabolites (i.e., antibiotics) not identified in this work that made this strain the best in vivo BCO against both pathogens.
Recent results obtained by Tian et al. (2022) [13] and by Yin et al. (2021) [42] show how different species of Bacillus (B. velezensis or B. cereus) could enhance resistance of cucumber to reduce M. incognita infection by activating several defense-responsive genes. Similarly, Egan and Kakouli-Duarte (2022) [43] investigated the potential role of some Pseudomas strains in M. javanica suppression in tomato plants through a split-root system and deduced that Induced Systemic Resistance (ISR) was the major nematode control mechanism. The mechanism of action of P. aeruginosa AC17 could be the production of one or more extracellular compounds with nematicide and fungicide properties or by triggering the ISR response in strawberry plants.
Pseudomonas aeruginosa’s ability to degrade many compounds that are recalcitrant to other bacterial species makes this strain useful for bioremediation and other industries; thus, many strains are formulated under commercial bioproducts [32]. The biocontrol activities showed by P. aeruginosa AC17 against both strawberry pathogens (M. phaseolina and M. hapla) suggest that this bacterial strain could be an optimal candidate for biocontrol of the two main SBP in the strawberry growing area of southern Spain.

Author Contributions

Conceptualization, M.C., B.d.l.S. and M.T.; methodology, M.C., B.d.l.S., M.D.V. and M.T.; investigation, M.C., B.d.l.S., M.D.V. and M.T.; data curation, M.C., B.d.l.S. and M.T.; writing—original draft preparation, M.C., B.d.l.S. and M.T.; writing—review and editing, M.C., B.d.l.S. and M.T. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Andalusian Institute of Agricultural and Fisheries Research and Training (IFAPA) and the European Regional Development Fund grant number PP.AVA.AVA2019.034.

Data Availability Statement

The data presented in this study are available on request from the corresponding author. The data are not publicly available due its proprietary nature.

Conflicts of Interest

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

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Figure 1. Plant growth-related activities, expressed as a percentage of the total plant growth activities, measured in bacterial strains isolated from strawberry and blueberry rhizospheres.
Figure 1. Plant growth-related activities, expressed as a percentage of the total plant growth activities, measured in bacterial strains isolated from strawberry and blueberry rhizospheres.
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Figure 2. Biocontrol-related enzymatic activities, expressed as a percentage of the total enzymatic activities, measured in bacterial strains isolated from strawberry and blueberry rhizospheres.
Figure 2. Biocontrol-related enzymatic activities, expressed as a percentage of the total enzymatic activities, measured in bacterial strains isolated from strawberry and blueberry rhizospheres.
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Figure 3. M. phaseolina growth inhibition (%) by bacterial strains isolated from strawberry and blueberry rhizospheres. Error bars correspond to standard deviations. Different letters indicate significant differences among bacterial strains from pairwise comparisons using an alpha of 0.05 after a Kruskal–Wallis test.
Figure 3. M. phaseolina growth inhibition (%) by bacterial strains isolated from strawberry and blueberry rhizospheres. Error bars correspond to standard deviations. Different letters indicate significant differences among bacterial strains from pairwise comparisons using an alpha of 0.05 after a Kruskal–Wallis test.
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Table
Table 1. Treatments evaluated in pot trials for biocontrol of strawberry soil-borne diseases.
Table 1. Treatments evaluated in pot trials for biocontrol of strawberry soil-borne diseases.
Treatment CodeBCOSBP
Ø-ØØØ
Ø-FC37B. velezensis FC37Ø
Ø-Hvs8B. frigoritolerans Hvs8Ø
Ø-AC17P. aeruginosa AC17Ø
MH-ØØM. hapla
MH-FC37B. velezensis FC37M. hapla
MH-Hvs8B. frigoritolerans Hvs8M. hapla
MH-AC17P. aeruginosa AC17M. hapla
MP-ØØM. phaseolina
MP-FC37B. velezensis FC37M. phaseolina
MP-HVS8B. frigoritolerans Hvs8M. phaseolina
MP-AC17P. aeruginosa AC17M. phaseolina
BCO: Biocontrol organism. SBP: Soil-borne pathogen.
Table
Table 2. Phylogenetic affiliation of bacterial strains isolated from strawberry (FC codes) and blueberry (AC codes) rhizospheres at IFAPA El Cebollar (Huelva) Spain.
Table 2. Phylogenetic affiliation of bacterial strains isolated from strawberry (FC codes) and blueberry (AC codes) rhizospheres at IFAPA El Cebollar (Huelva) Spain.
Strain CodeRelated Species (blast/ncbl)Accession NumberSimilitude %
FC5.2Pseudomonas kribbensisOQ25587899.59
FC10.1Xanthomonas translucensOQ25586399.73
FC10.2Cupriavidus metallidurasOQ255864100
FC14Cytobacillus firmusOQ25586599.52
FC15Comamonas testosteroniOQ255866100
FC17Arthrobacter pascensOQ25586799.59
FC18Pararhizobium herbaeOQ25586899.7
FC20Pseudomonas brassicacearum subsp. neoaurantiacaOQ25586999.93
FC21.2Agrobacterium arsenijeviciiOQ255870100
FC25Xanthomonas translucensOQ25587199.73
FC28Pseudomonas flavescensOQ25587298.83
FC32Bacillus albusOQ255873100
FC34Arthrobacter humicolaOQ25587499.31
FC35Pseudomonas nitritireducensOQ25587599.86
FC36Arthrobacter pascensOQ25587699.45
FC37Bacillus velezensisOU487633*
FC39Raoultella ornithinolyticaOQ25587799.86
AC9.1Priestia aryabhattaiOQ255855100
AC11.2Bacillus altitudinisOQ255851100
AC14Mixta calidaOQ255852100
AC15Enterobacter quasiroggenkampiiOQ25585399.86
AC17Pseudomonas aeruginosaOQ255854100
ACH2Chryseobacterium cucumelisOQ25586099.93
ACH10Klebsiella variicola subsp. variicolaOQ25585699.73
ACH14Achromobacter veterisilvaerOQ25585799.93
ACH15Microbacterium arborescensOQ25585899.93
ACH16Bacillus velezensisOQ25585999.93
ACH25Enterobacter kobeiOQ25586199.93
ACH29Curtobacterium citreumOQ25586299.72
Hvs8Brevibacterium frigoritoleransOU487634*
* Previously identified [16].
Table
Table 3. Macrophomina phaseolina symptoms in plants inoculated with BCO.
Table 3. Macrophomina phaseolina symptoms in plants inoculated with BCO.
Treatment CodeDead Plants(%)Plant
Severity
Index		Crown
Incidence (%)		Crown
Severity
Index		Petiole
Colonization (%)		Root
Colonization (%)
MP-Ø12.5 ± 8.5 a2.9 ± 0.2 a75.0 ± 11.2 a1.6 ± 0.3 a75.0 ± 11.2 a75.0 ± 11.2 a
MP-Hvs818.8 ± 10.0 a2.0 ± 0.3 b18.8 ± 10.0 b0.8 ± 0.4 ab12.5 ± 8.5 b50.0 ± 12.9 a
MP-AC170.0 ± 0.0 b1.5 ± 0.1 b0.0 ± 0.0 b0.0 ± 0.0 b0.0 ± 0.0 b53.3 ± 13.3 a
MP-FC376.3 ± 6.1 a1.9 ± 0.2 b31.3 ± 11.9 b0.6 ± 0.3 b31.3 ± 11.9 b43.8 ± 12.8 a
BCO: Biocontrol organism. SBP: Soil-borne pathogen. Data are the mean ± standard error of 16 replicates (two trials × eight replicated plots). Values followed by the same letter within a column do not differ significantly according to HSD Tukey’s tests (p < 0.05).
Table
Table 4. Meloidogyne hapla symptoms in plants inoculated with BCO.
Table 4. Meloidogyne hapla symptoms in plants inoculated with BCO.
Treatment CodeGalling IndexRKN Per Pot (750 mL)
MH-Ø2.4 ± 0.1 a3033 ± 172 a
MH-Hvs82.2 ± 0.1 a2819 ± 214 a
MH-AC170.9 ± 0.9 b1518 ± 130 b
MH-FC372.3 ± 0.2 b2708 ± 121 a
BCO: Biocontrol organism. SBP: Soil-borne pathogen. Data are the mean ± standard error of 16 replicates (two trials × eight replicated plots). Values followed by the same letter within a column do not differ significantly according to HSD Tukey’s or Dunn’s tests (p < 0.05).
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Camacho, M.; de los Santos, B.; Vela, M.D.; Talavera, M. Use of Bacteria Isolated from Berry Rhizospheres as Biocontrol Agents for Charcoal Rot and Root-Knot Nematode Strawberry Diseases. Horticulturae 2023, 9, 346. https://doi.org/10.3390/horticulturae9030346

AMA Style

Camacho M, de los Santos B, Vela MD, Talavera M. Use of Bacteria Isolated from Berry Rhizospheres as Biocontrol Agents for Charcoal Rot and Root-Knot Nematode Strawberry Diseases. Horticulturae. 2023; 9(3):346. https://doi.org/10.3390/horticulturae9030346

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Camacho, María, Berta de los Santos, María Dolores Vela, and Miguel Talavera. 2023. "Use of Bacteria Isolated from Berry Rhizospheres as Biocontrol Agents for Charcoal Rot and Root-Knot Nematode Strawberry Diseases" Horticulturae 9, no. 3: 346. https://doi.org/10.3390/horticulturae9030346

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