Hitching a Ride in the Phyllosphere: Surfactant Production of Pseudomonas spp. Causes Co-swarming of Pantoea eucalypti 299R.

Here, we demonstrate the beneficial effect of surfactant-producing pseudomonads on Pantoea eucalypti 299R. We conducted a series of experiments in environments of increasing complexity. P. eucalypti 299R (Pe299R), and Pseudomonas sp. FF1 (Pff1) or Pe299R and surfactant-production deficient Pseudomonas sp. FF1::ΔviscB (Pff1ΔviscB) were co-inoculated in broth, on swarming agar plates, and on plants. In broth, there were no differences in the growth dynamics of Pe299R when growing in the presence of Pff1 or Pff1ΔviscB. By contrast, on swarming agar plates, Pe299R was able to co-swarm with Pff1 which led to a significant increase in Pe299R biomass compared to Pe299R growing with Pff1ΔviscB or in monoculture. Finally in planta, and using the single-cell bioreporter for reproductive success (CUSPER), we found a temporally distinct beneficial effect of Pff1 on co-inoculated Pe299R subpopulations that did not occur in the presence of Pff1ΔviscB. We tested three additional surfactant-producing pseudomonads and their respective surfactant knockout mutants on PE299R on swarming agar showing similar results. This led us to propose a model for the positive effect of surfactant production during leaf colonization. Our results indicate that co-motility might be common during leaf colonization and adds yet another facet to the already manyfold roles of surfactants.

The impact of different Saccharomyces cerevisiae strains on microbial composition and quality of Chinese rice wine fermentations.

Chinese rice wine (CRW) is a popular fermented product in China, with complicated microbial composition and flavour compounds. To reveal the effects of different strains of Saccharomyces cerevisiae (N85 and XZ11) on the microbial composition in the process of brewing, metagenomic sequencing approaches were carried out to explore the dynamic changes of bacteria and fungi. Furthermore, the volatile compounds and organic acids in CRW were identified by headspace solid phase microextraction (HS-SPME)/gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) at the end of the brewing. Our results indicated that different S. cerevisiae strains could influence microbial compositions and especially affected the growth of Lactobacillus brevis and Pantoea ananatis. The changes in the microbial community structure contributed to the remarkable difference in the content of lactic acid, esters, alcohols, and aldehydes. Moreover, functional network analysis provided insights into the biological correlations between microbial species and metabolic pathways, that is, Lactobacillus genus had negative effects on metabolic activities. This study expands the idea of improving the quality of CRW by controlling the microbiome.

Fluorescent Protein Expression as a Proxy for Bacterial Fitness in a High-Throughput Assay.

Bacterial growth is classically assessed by measuring the increases in optical density of pure cultures in shaken liquid media. Measuring growth using optical density has severe limitations when studying multistrain interactions, as it is not possible to measure the growth of individual strains within mixed cultures. Here, we demonstrated that constitutively expressed fluorescent proteins can be used to track the growth of individual strains in different liquid media. Fluorescence measurements were highly correlated with optical density measurements and cell counts. This allowed us to assess bacterial growth not only in pure cultures but also in mixed bacterial cultures and determine the impact of a competitor on a focal strain, thereby assessing relative fitness. Furthermore, we were able to track the growth of two different strains simultaneously by using fluorescent proteins with differential excitation and emission wavelengths. Bacterial densities measured by fluorescence yielded more consistent data between technical replicates than optical density measurements. Our setup employs fluorescence microplate readers that allow high throughput and replication. IMPORTANCE We expand on an important limitation of the concept of measuring bacterial growth, which is classically limited to one strain at a time. By adopting our approach, it is possible to measure the growth of several bacterial strains simultaneously with high temporal resolution and in a high-throughput manner. This is important to investigate bacterial interactions, such as competition and facilitation.

Application of inorganic phosphate limitation to efficient isoprene production in Pantoea ananatis.

AIMS: Establishment of an efficient isoprene fermentation process by adopting inorganic phosphate limitation as the trigger to direct metabolic flux to the isoprene synthetic pathway. METHODS AND RESULTS: We constructed isoprene-producing strains of Pantoea ananatis (a member of the Enterobacteriaceae family) by integrating a heterologous mevalonate pathway and a metabolic switch that senses external inorganic phosphate (Pi) levels. This metabolic switch enabled dual-phase isoprene production, where the initial cell growth phase under Pi-saturating conditions was uncoupled from the subsequent isoprene production phase under Pi-limiting conditions. In fed-batch fermentation using our best strain (SWITCH-PphoC/pIspSM) in a 1-l bioreactor, isoprene concentration in the off-gas was maintained between 300 and 460 ppm during the production phase and at 20 ppm during the cell growth phase, respectively. The strain SWITCH-PphoC/pIspSM produced totally 2·5 g l-1 of isoprene from glucose with a 1·8% volumetric yield in 48 h. CONCLUSIONS: This proof-of-concept study demonstrated that our Pi-dependent dual-phase production system using a P. ananatis strain as a producer has potential for industrial-scale isoprene fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: This Pi-dependent dual-phase fermentation process could be an attractive and economically viable option for the production of various commercially valuable isoprenoids.

Bioreactor production of 2,3-butanediol by Pantoea agglomerans using soybean hull acid hydrolysate as substrate.

Production of 2,3-butanediol (2,3-BD) by Pantoea agglomerans strain BL1 was investigated using soybean hull hydrolysate as substrate in batch reactors. The cultivation media consisted of a mixture of xylose, arabinose, and glucose, obtained from the hemicellulosic fraction of the soybean hull biomass. We evaluated the influence of oxygen supply, pH control, and media supplementation on the growth kinetics of the microorganism and on 2,3-BD production. P. agglomerans BL1 was able to simultaneously metabolize all three monosaccharides present in the broth, with average conversions of 75% after 48 h of cultivation. The influence of aeration conditions employed demonstrated the mixed acid pathway of 2,3-BD formation by enterobacteria. Under fully aerated conditions (2 vvm of air), up to 14.02 g L-1 of 2.3-BD in 12 h of cultivation were produced, corresponding to yields of 0.53 g g-1 and a productivity of 1.17 g L-1 h-1, the best results achieved. These results suggest the production potential of 2,3-BD by P. agglomerans BL1, which has been recently isolated from an environmental consortium. The present work proposes a solution for the usage of the hemicellulosic fraction of agroindustry biomasses, carbohydrates whose utilization are not commonly addressed in bioprocess.

Plant species affects establishment of Escherichia coli O157:H7 gfp+ on leafy vegetables.

AIMS: Greenhouse trials were conducted with different cultivars of baby leaf spinach, rocket and Swiss chard and inoculation of Escherichia coli O157:H7 gfp+, to determine whether plant species and cultivar have an impact on the establishment of this strain. METHODS AND RESULTS: Three cultivars each of spinach, rocket and Swiss chard were spray inoculated with E. coli O157:H7 gfp+ at doses of log 7 CFU per ml. Due to the different lengths of growing period spinach and Swiss chard were spray inoculated three times and rocket five times, with final inoculation performed 3 days prior to harvest. After a growing period of 26-33 days, E. coli O157:H7 gfp+ was recovered from the leaf surface in mean populations between log 1 and 6 CFU per gram. The lowest occurrence of E. coli O157:H7 gfp+ was found on rocket leaves and the highest on spinach. There was no significant difference in the establishment of E. coli O157:H7 gfp+ between cultivars, but there were differences between plant species. Indigenous phyllosphere bacteria were pure cultured and identified with 16S rRNA gene sequencing. CONCLUSIONS: Despite the same high inoculation dose of E. coli O157:H7 gfp+ on leaves, the establishment rate differed between plant species. However, plant cultivar did not affect establishment. Pantoea agglomerans dominated the identified bacterial isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: As previous studies are inconclusive on choice of model plant species and cultivar, we studied whether plant species or cultivar determines the fate of E. coli O157:H7 gfp+ on leafy vegetables. The findings indicate that plant species is a key determinant in the establishment of E. coli O157:H7 gfp+.

Impacts of indoor surface finishes on bacterial viability.

Microbes in indoor environments are constantly being exposed to antimicrobial surface finishes. Many are rendered non-viable after spending extended periods of time under low-moisture, low-nutrient surface conditions, regardless of whether those surfaces have been amended with antimicrobial chemicals. However, some microorganisms remain viable even after prolonged exposure to these hostile conditions. Work with specific model pathogens makes it difficult to draw general conclusions about how chemical and physical properties of surfaces affect microbes. Here, we explore the survival of a synthetic community of non-model microorganisms isolated from built environments following exposure to three chemically and physically distinct surface finishes. Our findings demonstrated the differences in bacterial survival associated with three chemically and physically distinct materials. Alkaline clay surfaces select for an alkaliphilic bacterium, Kocuria rosea, whereas acidic mold-resistant paint favors Bacillus timonensis, a Gram-negative spore-forming bacterium that also survives on antimicrobial surfaces after 24 hours of exposure. Additionally, antibiotic-resistant Pantoea allii did not exhibit prolonged retention on antimicrobial surfaces. Our controlled microcosm experiment integrates measurement of indoor chemistry and microbiology to elucidate the complex biochemical interactions that influence the indoor microbiome.

Exploring the survival tactics and plant growth promising traits of root-associated bacterial strains under Cd and Pb stress: A modelling based approach.

The study represents a microbial method for reducing heavy metal stress in terrestrial environment. Two rhizobacterial strains Pantoea agglomerance (PC1) and Pseudomonas aeruginosa (SA) having the ability to tolerate Cd2+ and Pb2+ ions stress, were employed in this study. The growth promotion and survival tactics of the strains under metal stress were explored through kinetic growth model using logistic equation, Luedeking-Piret model and Box Behnken design. Study also involves the interaction of strains with Zea mays L. under Cd2+ and Pb2+ ions stress. Results revealed that both strains have the potential to tolerate 500 mg L-1 of Cd2+ and Pb2+, ions and maintained the plant growth promoting traits. The Luedeking-Piret model estimated the maximum value of IAA on biomass growth (YP/X) 5.377 µg g-1 and 10.3 µg g-1 under Cd2+ ions, while 7.742 µg g-1 and 18.071 µg g-1 under Pb2+ ions stress for strains SA and PC1, respectively. Further, phosphate solubilization activity was optimized with the help of response surface methodology using Box Behnken Design. The optimum solubilization by strain PC1 and SA was achieved at 100 and 150 mg L-1 of Cd2+, and 150 and 200 mg L-1 of Pb2+ ion concentration at the pH range 6.75 and 7.5 respectively. The interactive study with Zea mays L. showed significant increase in seed germination in the presence of Cd2+ and Pb2+ ions thereby proving them as potent plant growth promoters and metal stress reducing biological agents. Hence, the findings of the study suggest that rhizobacterial strains could be a sustainable tool for restoration of metal contaminated sites.

Selenium reduction by a defined co-culture of Shigella fergusonii strain TB42616 and Pantoea vagans strain EWB32213-2.

Selenium reduction was evaluated with pure batch cultures of Shigella fergusonii strain TB42616 (TB) and Pantoea vagans strain EWB32213-2 (EWB), respectively. A two-stage process, from Se(VI) to Se(IV) and then from Se(IV) to Se(0), was observed. The second stage of reduction, from Se(IV) to Se(0), was observed as the rate-limiting step resulting in accumulation of the more toxic Se(IV). In order to facilitate Se(VI) reduction and reduce Se(IV) accumulation, the Se(VI)-reducing strain TB was co-cultured with a Se(IV)-reducing strain EWB. Although Se(VI) reduction rate was not affected, Se(IV) reduction was significantly enhanced with low Se(IV) accumulation in the defined co-culture. Effects of culture composition as well as nitrate and arsenate on Se(VI) reduction were also investigated. A co-culture composition of 10:1 (EWB:TB) ratio was observed to achieve the best total selenium reduction. In addition, nitrate at 50 mg/L was observed to inhibit Se(IV) reduction but not Se(VI) reduction, while arsenate at 200 mg/L exhibited slight inhibition on both Se(VI) and Se(IV) reduction.

Interaction with Pantoea agglomerans Modulates Growth and Melanization of Sporothrix brasiliensis and Sporothrix schenckii.

Sporothrix brasiliensis and Sporothrix schenckii stand as the most virulent agents of sporotrichosis, a worldwide-distributed subcutaneous mycosis. The origin of Sporothrix virulence seems to be associated with fungal interactions with organisms living in the same environment. To assess this hypothesis, the growth of these two species in association with Pantoea agglomerans, a bacterium with a habitat similar to Sporothrix spp., was evaluated. Growth, melanization, and gene expression of the fungus were compared in the presence or absence of the bacterium in the same culture medium. Both S. brasiliensis and S. schenckii grew in contact with P. agglomerans yielding heavily melanized conidia after 5 days of incubation at 30 °C in Sabouraud agar. This increased melanin production occurred around bacterial colonies, suggesting that fungal melanization is triggered by a diffusible bacterial product, which is also supported by a similar pattern of melanin production during Sporothrix spp. growth in contact with heat-killed P. agglomerans. Growth of P. agglomerans was similar in the presence or absence of the fungus. However, the growth of S. brasiliensis and S. schenckii was initially inhibited, but further enhanced when these species were co-cultured with P. agglomerans. Moreover, fungi were able to use killed bacteria as both carbon and nitrogen sources for growth. Representational difference analysis identified overexpressed genes related to membrane transport when S. brasiliensis was co-cultured with the bacteria. The down-regulation of metabolism-related genes appears to be related to nutrient availability during bacterial exploitation. These findings can lead to a better knowledge on Sporothrix ecology and virulence.

Dracaena sanderiana endophytic bacteria interactions: Effect of endophyte inoculation on bisphenol A removal.

Bisphenol A (BPA) is one of the most abundant endocrine-disrupting compounds which is found in the aquatic environment. However, actual knowledge regarding the effect of plant-bacteria interactions on enhancing BPA removal is still lacking. In the present study, Dracaena sanderiana endophytic bacteria interactions were investigated to evaluate the effect of bacterial inoculation on BPA removal under hydroponic conditions. Two plant growth-promoting (PGP) bacterial strains, Bacillus thuringiensis and Pantoea dispersa, which have high BPA tolerance and can utilize BPA for growth, were used as plant inocula. P. dispersa-inoculated plants showed the highest BPA removal efficiency at 92.32 ±â€¯1.23% compared to other inoculated and non-inoculated plants. This was due to a higher population of the endophytic inoculum within the plant tissues which resulted in maintained levels of indole-3-acetic acid (IAA) for the plant's physiological needs and lower levels of reactive oxygen species (ROS). In contrast, B. thuringiensis-inoculated plants had a lower BPA removal efficiency. However, individual B. thuringiensis possessed a significantly higher BPA removal efficiency compared to P. dispersa. This study provides convincing evidence that not all PGP endophytic bacteria-plant interactions could improve the BPA removal efficiency. Different inocula and inoculation times should be investigated before using plant inoculation to enhance phytoremediation.

Indole enhances the survival of Pantoea ananatis YJ76 in face of starvation conditions.

Pantoea ananatis YJ76 is an indole-producing predominant diazotrophic endophyte isolated from rice having multiple growth-promoting effects on host plant. As a decomposition metabolite of L-tryptophan (L-Trp), indole is confirmed to regulate various physiological processes of bacteria. In this research, we found that indole significantly improves the survival of YJ76 in face of starvation conditions and the promoting effect is related to the glycogen accumulation promoted by indole, which is much more significant in the middle decline phase than in other growth phases. Since carbon storage regulator CsrA is a key inhibiting factor on the storage of glycogen in bacteria, we explored the relation between indole-enhanced glycogen accumulation and csrA expression and found that there is a positive correlation between indole-enhanced glycogen accumulation and the indole-inhibited csrA expression in YJ76, which implies the potential relation between CsrA regulation and indole regulatory pathway.

D-tyrosine affects aggregation behavior of Pantoea agglomerans.

D-amino acids have been proved to disassemble biofilms by disassociating the matrix. Pantoea agglomerans is characterized by the formation of another kind of multicellular structure called symplasmata, which also remains the ability to form biofilms. In this study, a rice diazotrophic endophyte P. agglomerans YS19 was selected as a model strain to explore the effects of D-amino acids on these two kinds of cell aggregate structures. It was discovered that D-tyrosine disassociates biofilm, yet promotes symplasmata formation. D-tyrosine showed no influence on bacterial growth yet promoted the bacterial motility and inhibited the expression of cellular MalE and OmpF proteins, which enriched our knowledge of the biological effect of D-amino acids and expanded the research ideas of symplasmata formation.

Perturbation of maize phenylpropanoid metabolism by an AvrE family type III effector from Pantoea stewartii.

AvrE family type III effector proteins share the ability to suppress host defenses, induce disease-associated cell death, and promote bacterial growth. However, despite widespread contributions to numerous bacterial diseases in agriculturally important plants, the mode of action of these effectors remains largely unknown. WtsE is an AvrE family member required for the ability of Pantoea stewartii ssp. stewartii (Pnss) to proliferate efficiently and cause wilt and leaf blight symptoms in maize (Zea mays) plants. Notably, when WtsE is delivered by a heterologous system into the leaf cells of susceptible maize seedlings, it alone produces water-soaked disease symptoms reminiscent of those produced by Pnss. Thus, WtsE is a pathogenicity and virulence factor in maize, and an Escherichia coli heterologous delivery system can be used to study the activity of WtsE in isolation from other factors produced by Pnss. Transcriptional profiling of maize revealed the effects of WtsE, including induction of genes involved in secondary metabolism and suppression of genes involved in photosynthesis. Targeted metabolite quantification revealed that WtsE perturbs maize metabolism, including the induction of coumaroyl tyramine. The ability of mutant WtsE derivatives to elicit transcriptional and metabolic changes in susceptible maize seedlings correlated with their ability to promote disease. Furthermore, chemical inhibitors that block metabolic flux into the phenylpropanoid pathways targeted by WtsE also disrupted the pathogenicity and virulence activity of WtsE. While numerous metabolites produced downstream of the shikimate pathway are known to promote plant defense, our results indicate that misregulated induction of phenylpropanoid metabolism also can be used to promote pathogen virulence.

Raman chemical imaging of the rhizosphere bacterium Pantoea sp. YR343 and its co-culture with Arabidopsis thaliana.

Chemical imaging of plant-bacteria co-cultures makes it possible to characterize bacterial populations and behaviors and their interactions with proximal organisms, under conditions closest to the environment in the rhizosphere. Here Raman micro-spectroscopy and confocal Raman imaging are used as minimally invasive probes to study the rhizosphere bacterial isolate, Pantoea sp. YR343, and its co-culture with model plant Arabidopsis thaliana by combining enhanced Raman spectroscopies with electron microscopy and principal component analysis (PCA). The presence of carotenoid pigments in the wild type Pantoea sp. YR343 was characterized using resonance Raman scattering, which was also used to confirm successful disruption of the crtB gene in an engineered carotenoid mutant strain. Other components of the Pantoea sp. YR343 cells were imaged in the presence of resonantly enhanced pigments using a combination of surface enhanced Raman imaging and PCA. Pantoea sp. YR343 cells decorated with Ag colloid synthesized ex situ gave spectra dominated by carotenoid scattering, whereas colloids synthesized in situ produced spectral signatures characteristic of flavins in the cell membrane. Scanning electron microscopy (SEM) of whole cells and transmission electron microscopy (TEM) images of thinly sliced cross-sections were used to assess structural integrity of the coated cells and to establish the origin of spectral signatures based on the position of Ag nanoparticles in the cells. Raman imaging was also used to characterize senescent green Arabidopsis thaliana plant roots inoculated with Pantoea sp. YR343, and PCA was used to distinguish spectral contributions from plant and bacterial cells, thereby establishing the potential of Raman imaging to visualize the distribution of rhizobacteria on plant roots.

A Large Repetitive RTX-Like Protein Mediates Water-Soaked Lesion Development, Leakage of Plant Cell Content and Host Colonization in the Pantoea stewartii subsp. stewartii Pathosystem.

Pantoea stewartii subsp. stewartii is the etiological agent of Stewart's wilt and is a serious bacterial pathogen affecting sweet corn. During the leaf blight phase, P. stewartii colonizes the leaf apoplast and causes a characteristic water-soaked lesion. The Hrp type III secretion system has been implicated in the water-soaking phenotype, and the goal of this study was to investigate other potential factors that contribute to the plant cellular disruption associated with these lesions. The P. stewartii genome contains a gene encoding a large repetitive RTX toxin, designated rtx2. RTX toxins comprise a large family of pore-forming proteins, which are widely distributed among gram-negative bacteria. These cytotoxins usually lyse their target host cells and cause significant tissue damage as a consequence. We hypothesized that this RTX-like toxin plays a role in the water-soaking phase of infection due to its predicted cytolytic properties. Based on the data reported here, we conclude that RTX2 contributes significantly to the development of water-soaked lesions and leakage of plant cellular contents and is an important pathogenicity factor for P. stewartii.

Cloning and characterization of 5-enopyruvylshikimate-3-phosphate synthase from Pantoea sp.

The shikimate pathway enzyme 5-enopyruvylshikimate-3-phosphate synthase (EPSPS) is the target of the broad spectrum herbicide glyphosate. A novel aroA gene encoding an EPSPS from Pantoea sp was identified and subcloned into the pET-28a vector to construct the recombinant pET-AroAPantoea sp plasmid. Amino acid sequence analysis indicated that AroAPantoea sp is a class I AroA enzyme. When expressed in Escherichia coli, it conveyed high tolerance to glyphosate. AroAPantoea sp may be used to generate transgenic glyphosate-tolerant plants.

AHL-type quorum sensing and its regulation on symplasmata formation in Pantoea agglomerans YS19.

Pantoea agglomerans YS19, an endophytic diazotrophic bacterium isolated from rice, is characterized by the formation of multicellular aggregate structure called symplasmata, which not only bestow the strong stress-resistance of the bacterium, but also contribute to the specific adaptation in the endophyte-host association. Acyl-homoserine lactones (AHLs), as the important signal molecule in the quorum sensing (QS) system of gram-negative bacteria, were demonstrated to regulate motility, cell-aggregation, and other bacterial behaviors. Here, the production of AHL by P. agglomerans YS19 and its regulation on the symplasmata formation were studied. It was revealed that the production of AHL by YS19 was initiated at the exponential growth stage and from then on, reached the peak values at the stationary growth stage in LB medium. The AHL was identified as N-3-oxooctanoyl-L-homoserine lactone (OOHL) by MALDI-TOF-MS analysis. The AHL synthesis gene pagI and receptor gene pagR in YS19 were cloned and phylogenetic analysis showed that they were high conservative among strains in species of P. agglomerans. It was revealed that AHL promoted the bacterial growth and symplasmata formation of YS19. Meanwhile, the colonization ability and growth-promoting effect of YS19 on the host plant were also enhanced by AHL. These results strongly suggest the pleiotropic effects of the AHL-type QS system in endophytic life of the strain.

[THE SENSITIVITY OF PHYTOPATHOGENIC BACTERIA TO STREPTOMYCIN UNDER THE INFLUENCE OF PESTICIDES].

The results of the streptomycin sensitivity changes of phytopathogenic Pseudomonas syringae and Xanthomonas translucens bacteria under the action of pesticides are pre- sented. It is demonstrated that phytopathogenic strains show greater changes of strepto- mycin sensitivity compared to epiphytic Pantoea agglomerans strain under the pesticides influence. Granstar herbicide, Tviks and Alpha Super insecticides increase the number of streptomycin resistant cells of Xanthomonas translucens 3164, P syringae pv. syringae YKM B-1027 and P syringae pv. atrofaciens YKM B-1011. This fact indicates mutagenic action of these pesticides against researched phytopathogenic bacteria.

Colonization of Madagascar periwinkle (Catharanthus roseus), by endophytes encoding gfp marker.

This study reports the introduction of gfp marker in two endophytic bacterial strains (Pantoea agglomerans C33.1, isolated from cocoa, and Enterobacter cloacae PR2/7, isolated from citrus) to monitor the colonization in Madagascar perinwinkle (Catharanthus roseus). Stability of the plasmid encoding gfp was confirmed in vitro for at least 72 h of bacterial growth and after the colonization of tissues, under non-selective conditions. The colonization was observed using fluorescence microscopy and enumeration of culturable endophytes in inoculated perinwinkle plants that grew for 10 and 20 days. Gfp-expressing strains were re-isolated from the inner tissues of surface-sterilized roots and stems of inoculated plants, and the survival of the P. agglomerans C33:1gfp in plants 20 days after inoculation, even in the absence of selective pressure, suggests that is good colonizer. These results indicated that both gfp-tagged strains, especially P. agglomerans C33.1, may be useful tools to deliver enzymes or other proteins in plant.