Preparation and evaluation of Bacillus megaterium-alginate microcapsules for control of rice sheath blight disease

Bacillus megaterium encapsulated in calcium alginate microcapsules was prepared and tested for its efficacy against sheath blight disease of rice. In laboratory conditions, the aqueous suspension (1:100, v/v in potato dextrose agar) of the bacterial microcapsules (10¹⁰ spores/ml) inhibited mycelial growth of Rhizoctonia solani (>99 %) after the microcapsules were produced and stored for 12 months at room temperature (28 ± 2 °C). The survival of the bacterium in the microcapsules in response to ultraviolet (u.v.) irradiation and high temperature was investigated. The survivability of the bacterium in the encapsulated form was greater than that of the fresh cells when it was subjected to u.v. (20-W General electric u.v. lamp from a 25 cm distance for 48 h) and a high temperature treatment (80 °C for 48 h). Cells of the bacterium were detected by scanning electron microscope on both the leaf sheath and the leaf blade (in pot tests in a greenhouse) after spraying encapsulated product. The number of bacteria on the surface of both rice tissues (5 Log. number/g of plant) after spraying with encapsulated product was not significantly different from that after spraying with fresh cells onto the rice seedlings. Spraying the encapsulated B. megaterium on rice plants in the greenhouse was as effective as spraying a chemical fungicide for suppressing rice sheath blight disease.     

Characterization of phosphate-solubilizing bacteria exhibiting the potential for growth promotion and phosphorus nutrition improvement in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) in calcareous soils of Sinaloa,Mexico

Greenhouse bioassays were used to examine the ability of selected strains of the rhizobacteria Sinorhizobium meliloti, Bacillus flexus and B. megaterium to solubilize phosphorus (P) and to affect growth promotion and phosphorus nutrition in maize. These bacterial strains were found to decrease the pH and solubilize some forms of insoluble P, such as tricalcium phosphate and hydroxyapatite, as well as to exhibit acid and alkaline phosphatase enzymatic activities in culture medium, properties that are possibly involved in P solubilization. Inoculation of the strains separately and as a consortium of the three bacteria (S. meliloti, B. flexus and B. megaterium) in P-deficient soil (4.33 w/v P) fertilized without P improved plant height, shoot and root dry weight, as well as P nutrition in the maize plants. Use of the B. flexus and B. megaterium strains separately and in a consortium positively affected several growth parameters and P nutrition in plants supplemented with insoluble P. No effect was observed when pots in which the seedlings were growing were supplied with soluble fertilizer. A second assay using a P-deficient soil (6.64 w/v P) showed that inoculation with the consortium of B. flexus and B. megaterium significantly increased growth and total P content in maize plants. A dose–response P fertilization experiment using sterile P-deficient soil led us to conclude that inoculation to soil of the mixture of B. flexus and B. megaterium may improve P nutrition and growth to a level previously attained by the addition of soluble P-fertilizer at 40 w/v P. A non-sterile experiment showed a beneficial response with B. megaterium but not with B. flexus. We propose utilizing these bacteria in P-deficient alkaline soils in future field trials in order to evaluate their potential as biofertilizers.     

Biocalcification by halophilic bacteria for remediation of concrete structures in marine environment

Microbial carbonate precipitation has emerged as a promising technology for remediation and restoration of concrete structures. Deterioration of reinforced concrete structures in marine environments is a major concern due to chloride-induced corrosion. In the current study, halophilic bacteria Exiguobacterium mexicanum was isolated from sea water and tested for biomineralization potential under different salt stress conditions. The growth, urease and carbonic anhydrase production significantly increased under salt stress conditions. Maximum calcium carbonate precipitation was recorded at 5 % NaCl concentration. Application of E. mexicanum on concrete specimens significantly increased the compressive strength (23.5 %) and reduced water absorption about five times under 5 % salt stress conditions compared to control specimens. SEM and XRD analysis of bacterial-treated concrete specimens confirmed the precipitation of calcite. The present study results support the potential of this technology for improving the strength and durability properties of building structures in marine environments.     

Halotolerant, alkaliphilic urease-producing bacteria from different climate zones and their application for biocementation of sand

Microbially induced calcium carbonate precipitation (MICP) is a phenomenon based on urease activity of halotolerant and alkaliphilic microorganisms that can be used for the soil bioclogging and biocementation in geotechnical engineering. However, enrichment cultures produced from indigenous soil bacteria cannot be used for large-scale MICP because their urease activity decreased with the rate about 5 % per one generation. To ensure stability of urease activity in biocement, halotolerant and alkaliphilic strains of urease-producing bacteria for soil biocementation were isolated from either sandy soil or high salinity water in different climate zones. The strain Bacillus sp. VUK5, isolated from soil in Ukraine (continental climate), was phylogenetically close in identity (99 % of 16S rRNA gene sequence) to the strain of Bacillus sp. VS1 isolated from beach sand in Singapore (tropical rainforest climate), as well as to the strains of Bacillus sp. isolated by other researchers in Ghent, Belgium (maritime temperate climate) and Yogyakarta, Indonesia (tropical rainforest climate). Both strains Bacillus sp. VS1 and VUK5 had maximum specific growth rate of 0.09/h and maximum urease activities of 6.2 and 8.8 mM of hydrolysed urea/min, respectively. The halotolerant and alkaliphilic strain of urease-producing bacteria isolated from water of the saline lake Dead Sea in Jordan was presented by Gram-positive cocci close to the species Staphylococcus succinus. However, the strains of this species could be hemolytic and toxigenic, therefore only representatives of alkaliphilic Bacillus sp. were used for the biocementation studies. Unconfined compressive strengths for dry biocemented sand samples after six batch treatments with strains VS1and VUK5 were 765 and 845 kPa, respectively. The content of precipitated calcium and the strength of dry biocemented sand at permeability equals to 1 % of initial value were 12.4 g Ca/kg of dry sand and 454 kPa, respectively, in case of biocementation by the strain VS1. So, halotolerant, alkaliphilic, urease-producing bacteria isolated from different climate zones have similar properties and can be used for biocementation of soil.     

Isolation of Arsenic-Resistant Bacteria from Bengal Delta Sediments and their Efficacy in Arsenic Removal from Soil in Association with Pteris vittata

The potential of arsenic-resistant bacteria in association with Pteris vittata to reduce the level of arsenic from soil was studied. The physicochemical characteristics of contaminated paddy soil were analyzed, and 3 bacterial isolates amongst 11 were screened and were selected for further study. These three isolates were characterized by 16S rDNA sequencing and identified as Bacillus altitudinis Strain SS8 (KJ432582), Bacillus megaterium Strain SS9 (KJ432583) and Lysinibacillus sp. Strain SS11 (KJ432584). Of these, Lysinibacillus sp. Strain SS11 displayed arsenic tolerance of 3256 mg L^?1 for arsenate and 1136 mg L^?1 for arsenite. Additionally, it showed bioaccumulation capacity of 23.43 mg L^?1 for arsenate and 5.65 mg L^?1 for arsenite. It also showed resistance to other heavy metals, especially towards iron, copper and chromium. It was also observed that Pteris vittata was able to take up more arsenic and iron from soil in the presence of these bacterial strains than in their absence, leading to contaminant-free soil. Thus, this system appears to be an effective bioremediating process to remove arsenic from contaminated soil.     

Improving the Shear Strength of Quartz Sand using the Microbial Method

An increase in urban population and the reduced number of suitable lands for construction projects have necessitated the need for ground improvement methods with no environmentally detrimental effects. Microbial-induced calcite precipitation (MICP) is a relatively environmentally friendly method for soil regeneration. In the present paper, X-ray diffraction (XRD) and scanning electron microscopy (SEM) analyses were performed to investigate the type and method of cement formation in loose quartz sand with different densities stabilized using bacteria. The results showed that the highest content of calcium carbonate cement was produced in the sample with the lowest density. Moreover, after microbial stabilization, the shear strength measured in the direct shear test was increased in the specimen with a lower density. We observed an increase in shear strength from 0.63 kg/cm² before injection for loose sand (γ = 1.5 g/cm³) to 3.92 kg/cm² after injection. In addition, the effect of injection time was investigated and found that shear strength in the two-stage injection per day is greater than that in one- and three-stage injections per day. With prolonging the injection time from one to five days, shear strength was significantly increased from 2.07 to 4.54 kg/cm². Furthermore, prolonging the bacterial treatment period led to a significant increase in the produced carbonate cement and, consequently, enhanced soil shear strength.     

Two-helper-strain co-culture system: a novel method for enhancement of 2-keto-l-gulonic acid production

A novel two-helper-strain co-culture system (TSCS) was developed to enhance 2-keto-l-gulonic acid (2-KLG) productivity for vitamin C production. Bacillus megaterium and B. cereus (with a seeding culture ratio of 1:3, v/v), used as helper strains, increased the 2-KLG yield using Ketogulonigenium vulgare compared to the conventional one-helper-strain (either B. cereus or B. megaterium) co-culture system (OSCS). After 45 h cultivation, 2-KLG concentration in the TSCS (69 g l^?1) increased by 8.9 and 7 % over that of the OSCS (B. cereus: 63.4 g l^?1; B. megaterium: 64.5 g l^?1). The fermentation period of TSCS was 4 h shorter than that of OSCS (B. cereus). The increased cell numbers of K. vulgare stimulated by the two helper strains possibly explain the enhanced 2-KLG yield. The results imply that TSCS is a viable method for enhancing industrial production of 2-KLG.     

Assessment of functional and genetic diversity of aerobic endospore forming Bacilli from rhizospheric soil of Phyllanthus amarus L.

Fifty two aerobic and endospore forming Bacilli (AEFB) strains were recovered from rhizospheric soil of Phyllanthus amarus. Morphological, biochemical and molecular characterization by 16S rDNA gene sequencing has shown that these bacterial strains belong to six different genera of AEFB i.e. Bacillus, Brevibacillus, Lysinibacillus, Paenibacillus, Terribacillus and Jeotgalibacillus. Analysis of their PGP activities has shown that 92.30 % strains produced indole acetic acid hormone, 86.53 % of the strains solubilized Phosphate and 44.23 % strains produced siderophore. Chitinase production activity was shown by 42.30 % of the strains and 21.15 % of the strains produced 1-amino cyclopropane-1-carboxylate (ACC) deaminase. 46.15 % of isolates have shown antagonistic activity against common fungal pathogen of the plant i.e. Corynespora cassiicola. Among all of the isolated strains B. Cereus JP44SK22 and JP44SK42 have shown all of the six plant growth promoting traits tested. B. megaterium strains (JP44SK18 and JP44SK35), Lysinibacillus sphaericus strains (JP44SK3 and JP44SK4) and Brevibacillus laterosporus strain JP44SK51 have also shown multiple PGP activities except ACC deaminase production activity. In the present study bacterial strain belonging to genera Jeotgalibacillus sp. JP44SK37 has been reported first time as a member of rhizospheric soil habitat and has also shown PGP activities. It can be concluded that Rhizosphere of P. amarus has harboured a good diversity of AEFB bacterial strains having a lot of biofertilizing and biocontrol abilities.     

Assessment of a Biostimulated or Bioaugmented Calcification System with Bacillus pasteurii in a Simulated Soil Environment

To evaluate how native soil microorganism and nutrients interact in a bioaugmented or biostimulated calcification process, batch experiments were conducted in saturated soil extract either amended with Bacillus pasteurii and/or NB-NH₄Cl media (nutrient broth, 3.0 g/L; NH₄Cl, 10.0 g/L; NaHCO₃, 2.12 g/L). The NB-NH₄Cl medium was chosen out of three potential candidates, where B. pasteurii precipitated the maximum amount of calcium (>95 %) as calcite in 12 h of incubation. The addition of B. pasteurii into saturated soil extract marginally improved the calcification to 4.26 % compared to the unamended control (3.2 %). Calcification with B. pasteurii in autoclaved soil extract was even better (18.75 %). However, the addition of the NB-NH₄Cl medium into the soil extract could significantly improve the calcification irrespective of whether B. pasteurii was added (88.46 %) or not (77.04 %). It suggests that soil microbial activity was not limiting even though soil nutrient was inadequate. It also indicates a possible negative interaction between soil microorganisms and B. pasteurii under nutrient-limited conditions.     

Plant growth promoting bacteria from Crocus sativus rhizosphere

Present study deals with the isolation of rhizobacteria and selection of plant growth promoting bacteria from Crocus sativus (Saffron) rhizosphere during its flowering period (October–November). Bacterial load was compared between rhizosphere and bulk soil by counting CFU/gm of roots and soil respectively, and was found to be ~40 times more in rhizosphere. In total 100 bacterial isolates were selected randomly from rhizosphere and bulk soil (50 each) and screened for in-vitro and in vivo plant growth promoting properties. The randomly isolated bacteria were identified by microscopy, biochemical tests and sequence homology of V1–V3 region of 16S rRNA gene. Polyphasic identification categorized Saffron rhizobacteria and bulk soil bacteria into sixteen different bacterial species with Bacillus aryabhattai (WRF5-rhizosphere; WBF3, WBF4A and WBF4B-bulk soil) common to both rhizosphere as well as bulk soil. Pseudomonas sp. in rhizosphere and Bacillus and Brevibacterium sp. in the bulk soil were the predominant genera respectively. The isolated rhizobacteria were screened for plant growth promotion activity like phosphate solubilization, siderophore and indole acetic acid production. 50 % produced siderophore and 33 % were able to solubilize phosphate whereas all the rhizobacterial isolates produced indole acetic acid. The six potential PGPR showing in vitro activities were used in pot trial to check their efficacy in vivo. These bacteria consortia demonstrated in vivo PGP activity and can be used as PGPR in Saffron as biofertilizers.This is the first report on the isolation of rhizobacteria from the Saffron rhizosphere, screening for plant growth promoting bacteria and their effect on the growth of Saffron plant.     

Optimization of growth medium for <Emphasis Type="Italic">Sporosarcina pasteurii</Emphasis> in bio-based cement pastes to mitigate delay in hydration kinetics

Microbial-induced calcium carbonate precipitation has been identified as a novel method to improve durability and remediate cracks in concrete. One way to introduce microorganisms to concrete is by replacing the mixing water with a bacterial culture in nutrient medium. In the literature, yeast extract often has been used as a carbon source for this application; however, severe retardation of hydration kinetics has been observed when yeast extract is added to cement. This study investigates the suitability of alternative carbon sources to replace yeast extract for microbial-induced calcium carbonate precipitation in cement-based materials. A combination of meat extract and sodium acetate was identified as a suitable replacement in growth medium for Sporosarcina pasteurii; this alternative growth medium reduced retardation by 75 % (as compared to yeast extract) without compromising bacterial growth, urea hydrolysis, cell zeta potential, and ability to promote calcium carbonate formation.     

Distribution of Bacillus thuringiensis in the complex of spore-forming bacteria of the genus Bacillus Cohn in the soils of the nut-fruit forests of southern Kyrgyzstan

Three-year-long investigation of the microflora in the soil of nut-fruit forests in the south of Kyrgyzstan showed a wide distribution of spore-forming bacteria Bacillus subtilis, B. cereus, B. idosus, and B. megaterium. In the complex of these bacteria, crystal-forming bacteria B. thuringiensis are abundant. The occurrence of B. thuringiensis in mountain-forest black-brown soils reaches 80% of soil samples. Dominating subspecies are tohokuensis (H17), israelensis (H14), and toguchini (H31). Abundance of B. thuringiensis was insignificant and accounted for only 1% of spore-forming bacteria. The abundance B. thuringiensis in oozy biocenoses on the shores of water bodies within the Sary-Chelek Biosphere Reserve reaches 12.6–18.0% of soil-forming bacteria. Thus, it is possible to assume that B. thuringiensis not only are conserved in soils but also can reproduce.     

Atmospheric methane removal by methane-oxidizing bacteria immobilized on porous building materials

Biological treatment using methane-oxidizing bacteria (MOB) immobilized on six porous carrier materials have been used to mitigate methane emission. Experiments were performed with different MOB inoculated in building materials at high (~20 % (v/v)) and low (~100 ppmv) methane mixing ratios. Methylocystis parvus in autoclaved aerated concrete (AAC) exhibited the highest methane removal rate at high (28.5?±?3.8 μg CH₄ g^?1 building material h^?1) and low (1.7?±?0.4 μg CH₄ g^?1 building material h^?1) methane mixing ratio. Due to the higher volume of pores with diameter >5 μm compared to other materials tested, AAC was able to adsorb more bacteria which might explain for the higher methane removal observed. The total methane and carbon dioxide-carbon in the headspace was decreased for 65.2?±?10.9 % when M. parvus in Ytong was incubated for 100 h. This study showed that immobilized MOB on building materials could be used to remove methane from the air and also act as carbon sink.     

High yield production of extracellular recombinant levansucrase by Bacillus megaterium

In this study, a high yield production bioprocess with recombinant Bacillus megaterium for the production of the extracellular enzyme levansucrase (SacB) was developed. For basic optimization of culture parameters and nutrients, a recombinant B. megaterium reporter strain that produced green fluorescent protein under control of a vector-based xylose-inducible promoter was used. It enabled efficient microtiter plate-based screening via fluorescence analysis. A pH value of pH?6, 20 % of dissolved oxygen, 37 °C, and elevated levels of biotin (100 μg?L^?1) were found optimal with regard to high protein yield and reduced overflow metabolism. Among the different compounds tested, fructose and glycerol were identified as the preferred source of carbon. Subsequently, the settings were transferred to a B. megaterium strain recombinantly producing levansucrase SacB based on the plasmid-located xylose-inducible expression system. In shake flask culture under the optimized conditions, the novel strain already secreted the target enzyme in high amounts (14 U?mL^?1 on fructose and 17.2 U?mL^?1 on glycerol). This was further increased in high cell density fed-batch processes up to 55 U?mL^?1, reflecting a levansucrase concentration of 0.52 g?L^?1. This is 100-fold more than previous efforts for this enzyme in B. megaterium and more than 10-fold higher than reported values of other extracellular protein produced in this microorganism so far. The recombinant strain could also handle raw glycerol from biodiesel industry which provided the same amount and quality of the recombinant protein and suggests future implementation into existing biorefinery concepts.     

Calcium carbonate in termite galleries – biomineralization or upward transport?

Termites and soil calcium carbonate are major factors in the global carbon cycle: termites by their role in decomposition of organic matter and methane production, and soil calcium carbonate by its storage of atmospheric carbon dioxide. In arid and semiarid soils, these two factors potentially come together by means of biomineralization of calcium carbonate by termites. In this study, we evaluated this possibility by testing two hypotheses. Hypothesis 1 states that termites biomineralize calcium carbonate internally and use it as a cementing agent for building aboveground galleries. Hypothesis 2 states that termites transport calcium carbonate particles from subsoil horizons to aboveground termite galleries where the carbonate detritus becomes part of the gallery construction. These hypotheses were tested by using (1) field documentation that determined if carbonate-containing galleries only occurred on soils containing calcic horizons, (2) ¹³C/¹²C ratios, (3) X-ray diffraction, (4) petrographic thin sections, (5) scanning electron microscopy, and (6) X-ray mapping. Four study sites were evaluated: a C₄-grassland site with no calcic horizons in the underlying soil, a C₄-grassland site with calcic horizons, a C₃-shrubland site with no calcic horizons, and a C₃-shrubland site with calcic horizons. The results revealed that carbonate is not ubiquitously present in termite galleries. It only occurs in galleries if subsoil carbonate exists within a depth of 100 cm. ¹³C/¹²C ratios of carbonate in termite galleries typically matched ¹³C/¹²C ratios of subsoil carbonate. X-ray diffraction revealed that the carbonate mineralogy is calcite in all galleries, in all soils, and in the termites themselves. Thin sections, scanning electron microscopy, and X-ray mapping revealed that carbonate exists in the termite gut along with other soil particles and plant opal. Each test argued against the biomineralization hypothesis and for the upward-transport hypothesis. We conclude, therefore, that the gallery carbonate originated from upward transport and that this CaCO₃ plays a less active role in short-term carbon sequestration than it would have otherwise played if it had been biomineralized directly by the termites.     

Production of single chain Fab (scFab) fragments in Bacillus megaterium

Background

The demand on antigen binding reagents in research, diagnostics and therapy raises questions for novel antibody formats as well as appropriate production systems. Recently, the novel single chain Fab (scFab) antibody format combining properties of single chain Fv (scFv) and Fab fragments was produced in the Gram-negative bacterium Escherichia coli. In this study we evaluated the Gram-positive bacterium Bacillus megaterium for the recombinant production of scFab and scFvs in comparison to E. coli.

Results

The lysozyme specific D1.3 scFab was produced in B. megaterium and E. coli. The total yield of the scFab after purification obtained from the periplasmic fraction and culture supernatant of E. coli was slightly higher than that obtained from culture supernatant of B. megaterium. However, the yield of functional scFab determined by analyzing the antigen binding activity was equally in both production systems. Furthermore, a scFv fragment with specificity for the human C reactive protein was produced in B. megaterium. The total yield of the anti-CRP scFv produced in B. megaterium was slightly lower compared to E. coli, whereas the specific activity of the purified scFvs produced in B. megaterium was higher compared to E. coli.

Conclusion

B. megaterium allows the secretory production of antibody fragments including the novel scFab antibody format. The yield and quality of functional antibody fragment is comparable to the periplasmic production in E. coli.     

Biosynthesis,characterization and antimicrobial activity of silver nanoparticles by Streptomyces sp. SS2

In the present study the microbial biosynthesis of silver nanoparticles (AgNPs) by secondary metabolites of Streptomyces sp. SS2 in an eco-friendly approach has been reported. The Streptomyces sp. SS2 was isolated from the soil sediment of Similipal Biosphere Reserve. The identification of this strain was determined by phenotypical characteristics (morphological and biochemical) and molecular characterization method using 16 s rDNA sequencing. The morphological study was also done by high-resolution scanning electron microscopy. The preliminary characterization of biosynthesized silver nanoparticle was carried out using UV–Vis spectrum analysis, which showed an absorption peak at 420 nm corresponding to plasmon absorption of silver. The average size and charge (zeta potential) of the particles were found to be 67.95 ± 18.52 nm and ?17.7 ± 5.30 mV, respectively. The functional groups were identified by FTIR studies and their morphology (round and spherical shape) was determined by scanning electron microscopy. The synthesized AgNPs exhibited excellent antibacterial activity against Escherichia coli (MTCC 1089), Bacillus subtilis (MTCC 7164), Staphylococcus epidermis (MTCC 3615), Vibrio cholerae (MTCC 3904) and Staphylococcus aureus (MTCC 1144). These biotechnological approaches of synthesis of nanoparticles can direct a new path in biomaterial sciences and enrich biomedical applications.     

Biocontrol and Plant Growth Promotion Characterization of Bacillus Species Isolated from Calendula officinalis Rhizosphere

The phenotypic and genotypic diversity of the plant growth promoting Bacillus genus have been widely investigated in the rhizosphere of various agricultural crops. However, to our knowledge this is the first report on the Bacillus species isolated from the rhizosphere of Calendula officinalis. 15 % of the isolated bacteria were screened for their important antifungal activity against Fusarium oxysporum, Botrytis cinerea, Aspergillus niger, Cladosporium cucumerinium and Alternaria alternata. The bacteria identification based on 16S r-RNA and gyrase-A genes analysis, revealed strains closely related to Bacillus amyloliquefaciens, B. velezensis, B. subtilis sub sp spizezenii and Paenibacillus polymyxa species. The electro-spray mass spectrometry coupled to liquid chromatography (ESI-LC MS) analysis showed that most of the Bacillus isolates produced the three lipopeptides families. However, the P. polymyxa (18SRTS) didn’t produce any type of lipopeptides. All the tested Bacillus isolates produced cellulase but the protease activity was observed only in the B. amyloliquefaciens species (9SRTS). The Salkowsky colorimetric test showed that the screened bacteria synthesized 6–52 μg/ml of indole 3 acetic acid. These bacteria produced siderophores with more than 10 mm wide orange zones on chromazurol S. The greenhouse experiment using a naturally infested soil with Sclerotonia sclerotiorum showed that the B. amyloliquefaciens (9SRTS) had no significant (P > 0.05) effect on the pre-germination of the chickpea seeds. However, it increased the size of the chickpea plants and reduced the stem rot disease (P < 0.05).These results suggested that the Bacillus strains isolated in this work may be further used as bioinoculants to improve the production of C. officinalis and other crop systems.     

Natural recovery from Verticillium wilt in olive: can it be exploited in a control strategy?

Aims

Our objective was to evaluate if natural recovery may be exploited in disease control of Verticillium wilt in olive. Therefore, we evaluated the following: the incidence of natural recovery; the Verticillium dahliae viability within olive tissues over time and the effectiveness of soil solarization, calcium cyanamide and pollarding of trees at soil level in promoting natural recovery.

Methods

Three different experiments (A, B and C) were performed in commercial olive orchards planted with the highly susceptible cv. ‘Bella di Cerignola’ and infested with the non-defoliating V. dahliae pathotype.

Results

In experiment A, in the period 2010–2012, natural recovery occurred on 35 of 138 diseased trees (25 %); however, this recovery was transient and lasted between 3 months for 11 trees (8 %) and 21 months for one tree (0.7 %). V. dahliae tended to be inactivated in twigs within 1 or 2 years after symptom onset (experiment A). However, it was evident that V. dahliae was more abundant in larger (trunk and first- or second-order branches) versus thinner woody parts of olive trees (roots; experiment B). In the attempt to explore whether natural recovery could be further stimulated artificially, it was observed that soil solarization and soil application of calcium cyanamide were ineffective in promoting its occurrence. Tree pollarding at soil level induced a transient recovery, which lasted only 1 year (experiment C).

Conclusions

Based on our observations, natural recovery of susceptible olive from Verticillium wilt has a low impact on the disease epidemiology in the short-term only and cannot be effectively stimulated in practice by soil solarization, calcium cyanamide or tree pollarding.