Synergistic Activity of Rhamnolipid Biosurfactant and Nanoparticles Synthesized Using Fungal Origin Chitosan Against Phytopathogens

Phytopathogens pose severe implications in the quantity and quality of food production by instigating several diseases. Biocontrol strategies comprising the application of biomaterials have offered endless opportunities for sustainable agriculture. We explored multifarious potentials of rhamnolipid-BS (RH-BS: commercial), fungal chitosan (FCH), and FCH-derived nanoparticles (FCHNPs). The high-quality FCH was extracted from Cunninghamella echinulata NCIM 691 followed by the synthesis of FCHNPs. Both, FCH and FCHNPs were characterized by UV-visible spectroscopy, DLS, zeta potential, FTIR, SEM, and Nanoparticle Tracking Analysis (NTA). The commercial chitosan (CH) and synthesized chitosan nanoparticles (CHNPs) were used along with test compounds (FCH and FCHNPs). SEM analysis revealed the spherical shape of the nanomaterials (CHNPs and FCHNPs). NTA provided high-resolution visual validation of particle size distribution for CHNPs (256.33 ± 18.80 nm) and FCHNPs (144.33 ± 10.20 nm). The antibacterial and antifungal assays conducted for RH-BS, FCH, and FCHNPs were supportive to propose their efficacies against phytopathogens. The lower MIC of RH-BS (256 μg/ml) was observed than that of FCH and FCHNPs (>1,024 μg/ml) against Xanthomonas campestris NCIM 5028, whereas a combination study of RH-BS with FCHNPs showed a reduction in MIC up to 128 and 4 μg/ml, respectively, indicating their synergistic activity. The other combination of RH-BS with FCH resulted in an additive effect reducing MIC up to 128 and 256 μg/ml, respectively. Microdilution plate assay conducted for three test compounds demonstrated inhibition of fungi, FI: Fusarium moniliforme ITCC 191, FII: Fusarium moniliforme ITCC 4432, and FIII: Fusarium graminearum ITCC 5334 (at 0.015% and 0.020% concentration). Furthermore, potency of test compounds performed through the in vitro model (poisoned food technique) displayed dose-dependent (0.005%, 0.010%, 0.015%, and 0.020% w/v) antifungal activity. Moreover, RH-BS and FCHNPs inhibited spore germination (61–90%) of the same fungi. Our efforts toward utilizing the combination of RH-BS with FCHNPs are significant to develop eco-friendly, low cytotoxic formulations in future

Pairwise variation analysis between NFn and NFn+1 to determine the optimal number of genes for reliable normalization.

<p>Values below the 0.15 threshold mean that <i>n</i> genes might be sufficient.</p

Selection of reference genes for quantitative real-time RT-PCR assays in different morphological forms of dimorphic zygomycetous fungus <i>Benjaminiella poitrasii</i>

<div><p><i>Benjaminiella poitrasii</i>, a dimorphic non-pathogenic zygomycetous fungus, exhibits a morphological yeast (Y) to hypha (H) reversible transition in the vegetative phase, sporangiospores (S) in the asexual phase and zygospores (Z) in the sexual phase. To study the gene expression across these diverse morphological forms, suitable reference genes are required. In the present study, 13 genes <i>viz</i>. <i>ACT</i>, <i>18</i>S r<i>RNA</i>, <i>eEF1α</i>, <i>eEF-Tu</i>,<i>eIF-1A</i>, <i>Tub-α</i>, <i>Tub-b</i>, <i>Ubc</i>, <i>GAPDH</i>, <i>Try</i>, <i>WS-21</i>, <i>NADGDH</i> and <i>NADPGDH</i> were evaluated for their potential as a reference, particularly for studying gene expression during the Y-H reversible transition and also for other asexual and sexual life stages of <i>B</i>. <i>poitrasii</i>. Analysis of RT-qPCR data using geNorm, normFinder and BestKeeper software revealed that genes such as <i>Ubc</i>, <i>18S rRNA</i> and <i>WS-21</i> were expressed at constant levels in each given subset of RNA samples from all the morphological phases of <i>B</i>. <i>poitrasii</i>. Therefore, these reference genes can be used to elucidate the role of morpho-genes in <i>B</i>. <i>poitrasii</i>. Further, use of the two most stably expressed genes (<i>Ubc</i> and <i>WS-21</i>) to normalize the expression of the ornithine decarboxylase gene (Bp<i>odc</i>) in different morphological forms of <i>B</i>. <i>poitrasii</i>, generated more reliable results, indicating that our selection of reference genes was appropriate.</p></div

Distribution overview of expression of each candidate reference gene in absolute Ct values for RNA samples of different morphological forms in <i>B</i>. <i>poitrasii</i>.

<p>Samples 1, yeast cells grown in YPG (1%) medium for 12 h; Sample 2, yeast cells grown in YPG medium for 24h; Sample 3, yeast cells grown in YPG (0.1%) medium for 12 h; Sample 4, hyphae grown in YP medium for 12 h; Sample 5, hyphae grown in YP medium for 24 h; Sample 6, hyphae grown in YPG(0.1%) for 12 h.; Sample 7, 5 d old sporangiospores; Sample 8, 10 d old sporangiospores; Sample 9, 15 d old sporangiospores; Sample 10, 7 d old zygospores; Sample 11, 15 d old zygospores; Sample 12, 30 d old zygospores.</p

Specificity of primers used for RT-qPCR analysis in <i>B</i>. <i>poitrasii</i>.

<p><b>A)</b> Agarose gel electrophoresis and analysis of amplified products obtained from RT-qPCR. The electrophoresis was done on 2% agarose gel. The names of candidate reference genes are mentioned on top. The figures on left indicate the size of PCR amplicons in base pairs. B) Melting curves indicating single peaks for three representative genes. Arrow indicates the melting curve for the RT-qPCR reaction without template.</p

List of the candidate reference genes evaluated in <i>B</i>. <i>poitrasii</i> and sequences of primers along with optimized annealing temperatures, size of the fragments they amplified and PCR efficiency.

<p>List of the candidate reference genes evaluated in <i>B</i>. <i>poitrasii</i> and sequences of primers along with optimized annealing temperatures, size of the fragments they amplified and PCR efficiency.</p

Comparison of expression stability of reference genes in different phases of <i>Benjaminiella poitrasii</i> as determined by geNorm, BestKeeper and normFinder software packages.

<p>Comparison of expression stability of reference genes in different phases of <i>Benjaminiella poitrasii</i> as determined by geNorm, BestKeeper and normFinder software packages.</p

Protein, cAMP, and NAD(P)H contents of different morphological forms of <i>Benjaminiella poitrasii</i>.

<p>Protein, cAMP, and NAD(P)H contents of different morphological forms of <i>Benjaminiella poitrasii</i>.</p

geNorm—based ranking of the candidate reference genes during different morphological stages of <i>B</i>. <i>poitrasii</i>.

<p>Genes were ranked from the least stable (on the left) to the most stable (on the right) according to their <i>M</i> value (Y axis). This classification was independently performed by using different sets of conditions. Vegetative stage, 6 samples of yeast and hyphal cells; Asexual stage, 3 samples of sporangiospores; Sexual stage, 3 samples of zygospores as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179454#pone.0179454.g002" target="_blank">Fig 2</a> legend.</p