Abstract
Late blight and wilt caused by the oomycete, Phytophthora infestans, and the fungus, Fusarium solani f. sp. eumartii, respectively, are severe diseases in Solanaceae crops worldwide. Although traditional approaches to control plant diseases have mainly relied on toxic chemical compounds, current studies are focused to identify more sustainable options. Finding alternatives, a low molecular weight chitosan (LMWCh) obtained from biomass of Argentine Sea’s crustaceans was assayed. In an attempt to characterize the action of LMWCh alone or in combination with the synthetic fungicide Mancozeb, the antimicrobial properties of LMWCh were assayed. In a side-by-side comparison with the SYTOX Green nucleic acid stain and the nitric oxide–specific probe, diaminofluorescein-FM diacetate (DAF-FM DA), yielded a similar tendency, revealing LMWCh-mediated cell death. The efficacy of LMWCh, Mancozeb, and the mixture LMWCh–Mancozeb was in turn tested. A synergistic effect in the reduction of F. eumartii spore germination was measured in the presence of subinhibitory dosis of 0.025 mg ml−1 LMWCh and 0.008 mg ml−1 Mancozeb. This mixture was efficient to increase the effectiveness of the single treatments in protecting against biotic stress judged by a drastic reduction of lesion area in P. infestans–inoculated tissues and activation of the potato defense responses.
Keywords
- chitosan
- Fusarium f. sp. Eumartii
- Mancozeb
- Phythophtora infestans
- potato
- tomato
1. Introduction
The Solanaceae species potato (
Despite the valuable contribution of Mancozeb to control plant diseases, alternatives and more sustainable options are still hot topics in phytopathology. In this sense, chitosan has been proved as a nontoxic and environmental-friendly compound for agricultural uses [5]. Chitosan is a linear polysaccharide composed of randomly distributed β-(1-4)-linked d-glucosamine and
This work provides data from
2. Materials and methods
2.1. Isolation and characterization of chitosan
Chitin was isolated from shells waste of the shrimp
2.2. Biological materials
2.3. Chemical treatments and inoculations
Commercial Mancozeb (800 g kg−1) WP (Dow AgroSciences, Argentina) was used at 8 mg ml−1 (8000 ppm) or at 0.08 mg ml−1 and 0.008 mg ml−1 in aqueous solution. Each solution was sprayed with a hand-held spray separately, or in combination with 0.25 mg ml−1 LMWCh on detached leaflets placed in Petri dishes. Controls were sprayed with distilled water. After being sprayed, leaflets were incubated at 18°C with light intensity of 120 µmol photons m−2 s−1 with 16:8 h light/dark cycles in a growth chamber. Each treatment combination was tested in a series of three independent trials.
After chemical treatments on detached leaflets, 10 µl of spore suspension
2.4. P. infestans sporangium germination assay
The sporangium germination assay was conducted as described by Mendieta et al. [19].
2.5. Measurements of F. eumartii spore germination
Determination of
2.6. Measurements of endogenous NO production in F. eumartii spores
Determination of endogenous NO production was monitored by incubating 107 spores ml−1 in 20 mM of HEPES-NaOH, pH 7.5, with different concentrations of LMWCh or LMWCh in combination with NO-specific scavenger, 2-(4-carboxyphenyl)-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO, 1 mM) as described in Terrile et al. [18].
2.7. Cell death stain and fluorescence microscopy
2.8. Protein extraction and western blot assay
Total proteins from potato leaflets were extracted as described by Terrile et al. [18]. Protein samples were boiled for 5 min and running on SDS-PAGE 12% polyacrylamide gels according to the method of Laemmli [20]. Proteins were transferred onto nitrocellulose using a semi-dry blotter (Invitrogen, USA) [21]. Immunodetection was performed using polyclonal antibodies raised against chitinase [22].
2.9. Statistical analysis
Treatments were established in a randomized complete block design, typically with four to seven treatments in each trial. The values shown in each figure are mean values ± SD of at least three experiments. Data were subjected to analysis of variance (one-way ANOVA), and
3. Results
3.1. LMWCh exerts antimicrobial action on P. infestans and F. eumartii .
The high water solubility and easy handling for agricultural application make LMWCh an attractive compound to deepen on its properties in the control of plant diseases. In this work, we hypothesized that LMWCh exerts protection against biotic stress in Solanaceae species, and in turn, it can be used in combination with reduced doses of Mancozeb (hundred times less than recommended dosage) for utilization in putative safer formulations. With the intention to move in that direction, we characterized the antimicrobial properties of LMWCh on
In turn, due to the easy handle and reproducibility,
SYTOX Green dye is often used to distinguish between live and dead cells. The fluorescence emission of SYTOX Green stain was measured in the presence of increasing concentrations of LMWCh. A substantial enhancement was measured in 0.25 and 2.5 mg ml−1 LMWCh-treated spores (Figure 3).
Then, LMWCh at 0.025 and 0.25 mg ml−1 and Mancozeb at 10−1 and 10−2 dilutions from the recommended field dosage were tested on fungal spore germination (Figure 4). Compared with control, the combination of suboptimal doses of 0.025 mg ml−1 LMWCh and 0.008 mg ml−1 Mancozeb had a remarkable inhibitory activity on spore germination (Figure 4). The treatment with the combined solution resulted in a synergistic effect in the reduction of spore germination. According to Limpel’s formula, the
3.2. LMWCh potentiates Mancozeb effect and protects against late blight in potato
The effect of LMWCh and Mancozeb treatments, alone or in combination, to control late blight in potato was tested. Meanwhile, single treatments revealed no significant differences, and the combined treatment of 0.25 mg ml−1 LMWCh and 0.08 mg ml−1 Mancozeb evidenced a reduction of at least sixfold in the late blight symptoms on potato leaflets (Figure 5).
With the aim to provide broader evidence on LMWCh-mediated mechanism, we assayed its elicitor properties in potato plants (Figure 6). Chitinases, the well-known pathogenesis-related proteins used as defense markers, are constitutively expressed at low levels, but highly induced by biotic stresses in potato leaves [22, 25]. Meanwhile, the 32 kDa chitinase was only detected at very low level in control; it moderately increased in Mancozeb-treated leaflets and was remarkably high in LMWCh-treated leaflets. Otherwise, the highest levels of chitinase isoforms were detected in potato leaflets upon LMWCh and Mancozeb treatment. Particularly, in the presence of LMWCh, the level of a third 27 kDa isoform was increased, suggesting that specific defense proteins are elicited by LMWCh in potato tissues (Figure 6).
4. Discussion
LMWCh was effective as an antimicrobial compound on spores from both
In this work, we demonstrated that the addition of LMWCh to an ineffective Mancozeb dose became a highly effective treatment to control late blight. The synergistic effect of LMWCh and commercially synthetic fungicides has been showed on
5. Conclusion
In summary, water-soluble LMWCh is an effective antimicrobial compound on spores from both
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