Carboxymethyl chitosan-N-alkylimine derivatives: Synthesis, characterization and use for preservation of symbiotic biofertilizer bacteria on chickpea seeds.

A series of carboxymethyl chitosan-N-alkylimine derivatives with side chain length of 4 to 10 carbons (CMCS-n, n = 4, 6, 8, 10) was prepared in a one-step solvent-free synthesis using Schiff base chemistry. The modified polysaccharides were characterized by their spectral, thermal and physical properties. The prepared polymers demonstrated an ability to spontaneous self-assembly with a clear correlation between critical aggregation concentration and the chain length of the alkyl substituent. N-alkylimine-CMCS derivatives were found to deliver hydrophobic (curcumin) and hydrophilic (ascorbic acid) active agents in unfavorable environments of water and oil, respectively. Then, N-alkylimine-CMCS derivatives were used as a platform for the delivery of symbiotic gram-positive bacteria Bacillus subtilis CJ onto chickpea seeds. These bacteria demonstrated a significantly higher survival rate (106 CFU/mL) in dried CMCS-6 derivative film than in other films tested. The seeds treated with N-alkylimine-CMCS coatings that contained B. subtilis CJ demonstrated up to 100-fold increase of this bacterial population on the seedlings in comparison to the pristine CMCS.

Seed-Derived Microbial Community of Wild Cicer Seedlings: Composition and Augmentation to Domesticated Cicer.

Seed-borne bacteria are a unique group of microorganisms capable of maintaining stable populations within plant tissues and seeds. These bacteria may benefit their host from germination to maturation and are of great interest for basic and applied plant-microbe interaction studies. Furthermore, many such beneficial bacteria present in wild plant species are missing in their respective congeneric domesticated forms. The objectives of this study were to explore the bacterial communities within the seeds of wild Cicer species and to select beneficial bacteria which could be used to improve production of domesticated chickpea (C. arietinum). We analyzed the composition of seed-borne bacteria of chickpea (Cicer spp.), comparing wild and domesticated species from different geographic locations. Subsequently, we isolated the dominant and prevalent seed-borne bacteria from wild Cicer judaicum and assessed their ability to colonize and affect the growth of domesticated chickpea and other legume crops. The composition and structure of seed-borne bacteria, determined by amplicon sequencing of the 16S rRNA gene, differed between wild and domesticated chickpea and varied among geographic locations. The genus Burkholderia dominated samples from domesticated chickpea at all examined sites, while Bacillus or Sphingomonas dominated cultures isolated from wild C. judaicum, dependent on geographic location. A particular Bacillus strain, Bacillus sp. CJ, representing the most prevalent bacterium in wild C. judaicum, was further isolated. Bacillus sp. CJ, applied by seed coating, successfully inhabited domesticated chickpea plants and improved plant growth parameters. These results demonstrate the potential for reconstructing the microbiota of crop plants using the wild microbiota reservoir. IMPORTANCE Chickpea (garbanzo bean, hummus, Cicer arietinum) representing the third legume crop produced globally. As is the case for many other domesticated crops, the adaptation and resistance of chickpea to biotic and abiotic stresses is inferior compared to that of their wild progenitors and relatives. Re-establishing desirable characteristics from wild to domesticated species may be achieved by reconstructing beneficial microbiota. In this study, we examined the seed-associated microbiota of both wild and domesticated chickpea and applied isolated beneficial bacteria originating from wild Cicer judaicum to domesticated chickpea by seed coating. This isolate, Bacillus sp. CJ, was successfully established in the crop and enhanced its growth, demonstrating effective and efficient manipulation of the chickpea microbiota as a potential model for future application in other crop plants.

Evaluation of the biocontrol activity of Frateuria defendens-derived metabolites against mollicutes.

Frateuria defendens is a candidate biocontrol agent that has been shown to reduce phytoplasma-related disease symptoms in grapevines and periwinkle plants. While a crude filtrate prepared from F. defendens can inhibit mollicute growth, the specific growth parameters for this bacterium, necessary to enhance this protective inhibitory response, remain unknown. Moreover, the separation of filtrate preparations from bacterial cells via centrifugation and filtration is laborious and time-consuming. As such, the present study was conducted to define the optimal growth conditions associated with maximal inhibitory activity of F. defendens and to establish a better approach to separating these bacterial cells from their secreted metabolites. To conduct these analyses, F. defendens was cultured in a range of media types, while associated inhibitory effects were tested in vitro using Spiroplasma melliferum as a model mollicute bacterium, and in planta using phytoplasma-infected periwinkle plantlets. These analyses revealed F. defendens growth patterns change based upon media composition, with filtrates prepared from a specific rich medium (S-medium) exhibiting beneficial activities, including the inhibition of S. melliferum and enhanced plant growth. When F. defendens cells were grown within semi-permeable, membrane-coated Small Bioreactor Platform (SBP) capsules, they could be more readily separated from the secreted metabolite fraction, obviating the need for filtration and/or centrifugation. This study is the first to have reported the use of SBP capsules to separate bacterial cells from their secreted metabolites under sterile conditions while retaining the ability of these metabolites to inhibit S. melliferum growth and to benefit the host plant. The results highlight promising new approaches to the effective biocontrol of phytoplasma-driven diseases in grapevines and other economically important plant species.

Characterization of an endophytic bacterium (Pseudomonas aeruginosa), originating from tomato (Solanum lycopersicum L.), and its ability to inhabit the parasitic weed Phelipanche aegyptiaca.

PHELIPANCHE AEGYPTIACA: is an obligate holo-parasitic weedlacking a functional photosynthetic system, which subsists on roots of a wide range of host crops, causing severe losses in yield quality and quantity. The parasite and its host are connected through their vascular system, forming a unique ecological system that enables the exchange of various substances. In a previous study, it was suggested that endophytic bacteria, which naturally inhabit the internal tissues of plants, can also be transmitted from the parasitic weed to its host and vice versa. In the current study, we investigate the characteristics of a previously isolated Pseudomonas sp. PhelS10 strain using both biochemical and molecular methods. This isolate was obtained from tomato plant tissue and was able to reduce P. aegyptiaca parasitism, and thus it may serve as a biocontrol agent. Our results revealed that production of Pseudomonas aeruginosa quinolone signal (PQS) was 2.1 times higher than that of the standard Pseudomonas aeruginosa strain (PAO1), which contributed to a 22% higher biofilm formation capability. PhelS10 strain was detected in the xylem of tomato plants using FISH analysis. In addition, PhelS10 strain was found in the parasitic weed's inner tissues, confirming the hypothesis that endophytic bacteria traffic between the host plant and its parasitic weed.

Antimicrobial Activity of Metabolites Secreted by the Endophytic Bacterium Frateuria defendens.

Candidatus Phytoplasma, the causative agent of yellows disease, inflicts substantial damage on several hundred plant species including perennials and annual plants. The endophytic bacterium Frateuria defendens reduces the symptoms of yellows disease in a number of agricultural crops. One possible mode of action is that the bacterium secretes antimicrobial metabolites. To test this hypothesis, the substances secreted by the endophyte during 10 days of growth in an artificial medium were identified by GC-MS (gas chromatography-mass spectrometry). Synthetic analogues to these substances were then used on periwinkle, a nurse culture plant infected by phytoplasma. Phytoplasma quantities were evaluated by quantitative PCR, and disease symptoms were monitored and recorded. It was found that specific compounds identified by the biochemical analysis caused a significant reduction in both the titer of phytoplasma and the disease symptoms in periwinkle when compared to untreated infected plants. Further research is required to examine the potential of these compounds as an effective treatment against yellows disease.

Frateuria defendens sp. nov., bacterium isolated from the yellows grapevine's disease vector Hyalesthes obsoletus.

A Dyella-like bacterium was previously isolated from the planthopper Hyalesthes obsoletus (Hemiptera). Based on its 16S rRNA gene sequence, strain DHoT was assigned to the family Rhodanobacteraceae with Dyella and Frateuria as its closest relatives. The closest 16S rRNA gene sequences were Frateuria aurantia DSM 6220T (98.2 %), Dyella thiooxydans ATSB10T (98 %), Dyella terrae JS14-6T (97.8 %) and Dyella marensis CS5-B2T (97.8 %). Strain DHoT is a Gram-negative, aerobic, motile, yellow-pigmented, rod-shaped bacterium. Strain DHoT cells grew well at 28-30 °C and at pH 6.5-7.5 on a nutrient agar plate. DNA-DNA hybridization showed that the relatedness between strain DHoT and D. jiangningensis strain SBZ3-12T, and F. aurantia DSM 6220T was 42.7 and 42.6 %, respectively. Ubiquinone Q-8 was the predominant respiratory quinone, and the major fatty acids (>10 %) were iso-C15 : 0, iso-C16 : 0 and iso-C17 : 0. In silico analysis based on phylogenetics and sequence identity at the nucleotide and protein levels suggests that Frateuria is the closest known relative of strain DHoT. Based on the phenotypic, chemotaxonomic and phylogenetic data, strain DHoT was designated as a novel species of the genus Frateuria, for which the name Frateuria defendens sp. nov. is proposed. The type strain is DHoT (=NCCB 100648T; =DLBT=DSM 106169T).

Introduction of a putative biocontrol agent into a range of phytoplasma- and liberibacter-susceptible crop plants.

BACKGROUND: Phytoplasma, the causative agent of Bois Noir disease of grapevines, are vectored by the planthopper Hyalesthes obsoletus (Hemiptera: Cixiidae). A Dyella-like bacterium (DLB) isolated from H. obsoletus inhibits the growth of Spiroplasma melliferum, a cultivable relative of phytoplasma. Additional evidence suggests that DLB can reduce the symptoms of yellows disease in grapevine plantlets. The aim of this study was to test whether DLB could colonize a range of phytoplasma- and liberibacter-sensitive crop plants, and thus assess its potential agricultural use. RESULTS: Vitex agnus-castus, the preferred host plant of H. obsoletus was found to be a natural host of DLB, which was successfully introduced into a range of crop plants belonging to seven families. The most effective DLB application method was foliar spraying. Microscopy observation revealed that DLB aggregated on the leaf surface and around the stomata, suggesting that this is its route of entry. DLB was also present in the vascular tissues of plants, indicating that it moved systemically through the plant. CONCLUSIONS: DLB is a potential biocontrol agent and its broad spectrum of host plants indicates the possibility of its future use against a range of diseases caused by phloem-limited bacteria. © 2017 Society of Chemical Industry.

Dyella-Like Bacterium Isolated from an Insect as a Potential Biocontrol Agent Against Grapevine Yellows.

Yellows diseases, caused by phytopathogenic bacteria of the genus Phytoplasma, are a major threat to grapevines worldwide. Because conventional applications against this pathogen are inefficient and disease management is highly challenging, the use of beneficial bacteria has been suggested as a biocontrol solution. A Dyella-like bacterium (DLB), isolated from the Israeli insect vector of grapevine yellows (Hyalesthes obsoletus), was suggested to be an endophyte. To test this hypothesis, the bacterium was introduced by spraying the plant leaves, and it had no apparent phytotoxicity to grapevine. Fluorescent in situ hybridization analysis showed that DLB is colonizing grapevine phloem. Because phytoplasmas inhabit the same niche, DLB interactions with this phytopathogen were examined. When the isolate was introduced to phytoplasma-infected Chardonnay plantlets, morphological disease symptoms were markedly reduced. The mode of DLB action was then tested using bioinformatics and system biology tools. DLB genome analysis suggested that the ability to reduce phytoplasma symptoms is related to inhibition of the pathogenic bacterium. These results provide the first step in examining the potential of DLB as a biological control agent against phytoplasmas in grapevine and, possibly, other agricultural crops.

An exceptional family: Ophiocordyceps-allied fungus dominates the microbiome of soft scale insects (Hemiptera: Sternorrhyncha: Coccidae).

Hemipteran insects of the suborder Sternorrhyncha are plant sap feeders, where each family is obligately associated with a specific bacterial endosymbiont that produces essential nutrients lacking in the sap. Coccidae (soft scale insects) is the only major sternorrhynchan family in which obligate symbiont(s) have not been identified. We studied the microbiota in seven species from this family from Israel, Spain and Cyprus, by high-throughput sequencing of ribosomal genes, and found that no specific bacterium was prevalent and abundant in all the tested species. In contrast, an Ophiocordyceps-allied fungus sp.-a lineage widely known as entomopathogenic-was highly prevalent. All individuals of all the tested species carried this fungus. Phylogenetic analyses showed that the Ophiocordyceps-allied fungus from the coccids is closely related to fungi described from other hemipterans, and they appear to be monophyletic, although the phylogenies of the Ophiocordyceps-allied fungi and their hosts do not appear to be congruent. Microscopic observations show that the fungal cells are lemon-shaped, are distributed throughout the host's body and are present in the eggs, suggesting vertical transmission. Taken together, the results suggest that the Ophiocordyceps-allied fungus may be a primary symbiont of Coccidae-a major evolutionary shift from bacteria to fungi in the Sternorrhyncha, and an important example of fungal evolutionary lifestyle switch.

Encapsulated Pseudomonas putida for phenol biodegradation: Use of a structural membrane for construction of a well-organized confined particle.

Phenols are toxic byproducts from a wide range of industry sectors. If not treated, they form effluents that are very hazardous to the environment. This study presents the use of a Pseudomonas putida F1 culture encapsulated within a confined environment particle as an efficient technique for phenol biodegradation. The innovative encapsulation technique method, named the "Small Bioreactor Platform" (SBP) technology, enables the use of a microfiltration membrane constructed as a physical barrier for creating a confined environment for the encapsulated culture. The phenol biodegradation rate of the encapsulated culture was compared to its suspended state in order to evaluate the effectiveness of the encapsulation technique for phenol biodegradation. A maximal phenol biodegradation rate (q) of 2.12/d was exhibited by encapsulated P. putida at an initial phenol concentration of 100 mg/L. The biodegradation rate decreased significantly at lower and higher initial phenol concentrations of 50 and up to 3000 mg/L, reaching a rate of 0.1018/d. The results also indicate similar and up to double the degradation rate between the two bacterial states (encapsulated vs. suspended). High resolution scanning electron microscopy images of the SBP capsule's membrane morphology demonstrated a highly porous microfiltration membrane. These results, together with the long-term activity of the SBP capsules and verification that the culture remains pure after 60 days using 16S rRNA gene phylogenetic affiliation tests, provide evidence for a successful application of this new encapsulation technique for bioaugmentation of selected microbial cultures in water treatment processes.

Host-Parasite-Bacteria Triangle: The Microbiome of the Parasitic Weed Phelipanche aegyptiaca and Tomato-Solanum lycopersicum (Mill.) as a Host.

Broomrapes (Phelipanche/Orobanche spp.) are holoparasitic plants that subsist on the roots of a variety of agricultural crops, establishing direct connections with the host vascular system. This connection allows for the exchange of various substances and a possible exchange of endophytic microorganisms that inhabit the internal tissues of both plants. To shed some light on bacterial interactions occurring between the parasitic Phelipanche aegyptiaca and its host tomato, we characterized the endophytic composition in the parasite during the parasitization process and ascertained if these changes were accompanied by changes to endophytes in the host root. Endophyte communities of the parasitic weed were significantly different from that of the non-parasitized tomato root but no significant differences were observed between the parasite and its host after parasitization, suggesting the occurrence of bacterial exchange between these two plants. Moreover, the P. aegyptiaca endophytic community composition showed a clear shift from gram negative to gram-positive bacteria at different developmental stages of the parasite life cycle. To examine possible functions of the endophytic bacteria in both the host and the parasite plants, a number of unique bacterial candidates were isolated and characterized. Results showed that a Pseudomonas strain PhelS10, originating from the tomato roots, suppressed approximately 80% of P. aegyptiaca seed germination and significantly reduced P. aegyptiaca parasitism. The information gleaned in the present study regarding the endophytic microbial communities in this unique ecological system of two plants connected by their vascular system, highlights the potential of exploiting alternative environmentally friendly approaches for parasitic weed control.

Bacterial associates of Hyalesthes obsoletus (Hemiptera: Cixiidae), the insect vector of bois noir disease, with a focus on cultivable bacteria.

The planthopper Hyalesthes obsoletus (Hemiptera: Cixiidae) is an important vector of phytoplasma diseases in grapevine. In the current study, the bacterial community compositions of symbionts of this insect were examined. Two dominant bacterial lineages were identified by mass sequencing: the obligate symbiont Candidatus Sulcia, and a facultative symbiont that is closely related to Pectobacterium sp. and to BEV, a cultivable symbiont of another phytoplasma vector, the leafhopper Euscelidius variegatus. In addition, one bacterium was successfully isolated in this study - a member of the family Xanthomonadaceae that is most closely related to the genus Dyella. This Dyella-like bacterium (DLB) was detected by FISH analysis in H. obsoletus guts but not ovaries, and its prevalence in H. obsoletus increased during the fall, suggesting that it was acquired by the host through feeding. We found that DLB inhibits Spiroplasma melliferum, a cultivable relative of phytoplasma, suggesting that it is a potential candidate for biological control against phytoplasma in grapevines.

Draft Genome Sequence of a Dyella-Like Bacterium from the Planthopper Hyalesthes obsoletus.

We report here the draft genome sequence of a Dyella-like bacterium (DLB) isolated from Hyalesthes obsoletus, the insect vector of the uncultivable mollicute bacterium "Candidatus Phytoplasma." This isolate inhibits Spiroplasma melliferum, a cultivable mollicute. The draft genome of DLB consists of 4,196,214 bp, with a 68.6% G+C content, and 3,757 genes were predicted.

Microbial associates of the vine mealybug Planococcus ficus (Hemiptera: Pseudococcidae) under different rearing conditions.

Sap-feeding insects harbor diverse microbial endosymbionts that play important roles in host ecology and evolution, including contributing to host pest status. The vine mealybug, Planococcus ficus, is a serious pest of grapevines, vectoring a number of pathogenic grape viruses. Previous studies have shown that virus transmission is abolished when mealybugs are raised in the laboratory on potato. To examine the possible role of microbial symbionts in virus transmission, the archaeal, bacterial, and fungal microbiota of field and laboratory P. ficus were characterized using molecular and classical microbiological methods. Lab and field colonies of P. ficus harbored different microbiota. While both were dominated by the bacterial obligate nutritional symbionts Moranella and Tremblaya, field samples also harbored a third bacterium that was allied with cluster L, a lineage of bacterial symbionts previously identified in aphids. Archaea were not found in any of the samples. Fungal communities in field-collected mealybugs were dominated by Metschnikowia and Cladosporium species, while those from laboratory-reared mealybugs were dominated by Alternaria and Cladosporium species. In conclusion, this study has identified a diverse set of microbes, most of which appear to be facultatively associated with P. ficus, depending on environmental conditions. The role of various members of the mealybug microbiome, as well as how the host plant affects microbial community structure, remains to be determined.

Bacterial endosymbionts in field-collected samples of Trialeurodes sp. nr. abutiloneus (Hemiptera: Aleyrodidae).

Facultative bacterial endosymbionts are common, influential associates of arthropods, yet their movement among host species has not been well documented. Plant-mediated transmission of Rickettsia has been shown for the whitefly Bemisia tabaci. Bemisia tabaci in USA cotton fields harbors the secondary symbionts Rickettsia and Hamiltonella, and co-occurs with Trialeurodes sp. nr. abutiloneus whiteflies. To determine whether symbionts may be shared, the microbial diversity of these whiteflies on cotton across the USA was analyzed. Trialeurodes sp. nr. abutiloneus bore Portiera, Pseudomonas, Serratia, Arsenophonus and Wolbachia. No Rickettsia or Hamiltonella were detected. These results provide no evidence for horizontal transmission of symbionts between these whitefly genera.

Novel Rickettsiella bacterium in the leafhopper Orosius albicinctus (Hemiptera: Cicadellidae).

Bacteria in the genus Rickettsiella (Coxiellaceae), which are mainly known as arthropod pathogens, are emerging as excellent models to study transitions between mutualism and pathogenicity. The current report characterizes a novel Rickettsiella found in the leafhopper Orosius albicinctus (Hemiptera: Cicadellidae), a major vector of phytoplasma diseases in Europe and Asia. Denaturing gradient gel electrophoresis (DGGE) and pyrosequencing were used to survey the main symbionts of O. albicinctus, revealing the obligate symbionts Sulcia and Nasuia, and the facultative symbionts Arsenophonus and Wolbachia, in addition to Rickettsiella. The leafhopper Rickettsiella is allied with bacteria found in ticks. Screening O. albicinctus from the field showed that Rickettsiella is highly prevalent, with over 60% of individuals infected. A stable Rickettsiella infection was maintained in a leafhopper laboratory colony for at least 10 generations, and fluorescence microscopy localized bacteria to accessory glands of the female reproductive tract, suggesting that the bacterium is vertically transmitted. Future studies will be needed to examine how Rickettsiella affects host fitess and its ability to vector phytopathogens.

Isolation and bioaugmentation of an estradiol-degrading bacterium and its integration into a mature biofilm.

Bioaugmentation can alter the potential activity as well as the composition of the naturally occurring microbial biota during bioremediation of a contaminated site. The focus of the current study is the pollutant 17ß-estradiol (E2), which can cause endocrine effects and is potentially harmful to aquatic biota and to public health. The community composition and function of biofilms, originating from a wetland system, as affected by augmentation of an estradiol-degrading bacterium (EDB-LI1) under different conditions, were investigated. EDB-LI1 inoculation into biofilm from two wetland ponds representing early and advanced water treatment stages, respectively, yielded three significant observations, as follows: (i) EDB-LI1, enriched from a biofilm of a constructed wetland wastewater treatment system, was detected (by quantitative PCR [qPCR] analysis) in this environment in the augmented biofilm only; (ii) the augmented biofilm acquired the ability to remove estradiol; and (iii) the bacterial community composition (analyzed by PCR-denaturing gradient gel electrophoresis [DGGE]) of the augmented biofilm differed from that of the control biofilm. Furthermore, EDB-LI1 bioaugmentation showed a higher level of removal of estradiol with biofilms that originated from the advanced-treatment-stage wetland pond than those from the early-treatment-stage pond. Hence, the bioaugmentation efficiency of EDB-LI1 depends on both the quality of the feed water and the microbial community composition in the pond.

The impact of reduced pH on the microbial community of the coral Acropora eurystoma.

Rising concentrations of atmospheric carbon dioxide are acidifying the world's oceans. Surface seawater pH is 0.1 units lower than pre-industrial values and is predicted to decrease by up to 0.4 units by the end of the century. This change in pH may result in changes in the physiology of ocean organisms, in particular, organisms that build their skeletons/shells from calcium carbonate, such as corals. This physiological change may also affect other members of the coral holobiont, for example, the microbial communities associated with the coral, which in turn may affect the coral physiology and health. In the present study, we examined changes in bacterial communities in the coral mucus, tissue and skeleton following exposure of the coral Acropora eurystoma to two different pH conditions: 7.3 and 8.2 (ambient seawater). The microbial community was different at the two pH values, as determined by denaturing gradient gel electrophoresis and 16S rRNA gene sequence analysis. Further analysis of the community in the corals maintained at the lower pH revealed an increase in bacteria associated with diseased and stressed corals, such as Vibrionaceae and Alteromonadaceae. In addition, an increase in the number of potential antibacterial activity was recorded among the bacteria isolated from the coral maintained at pH 7.3. Taken together, our findings highlight the impact that changes in the pH may have on the coral-associated bacterial community and their potential contribution to the coral host.

Microbial population and activity in wetland microcosms constructed for improving treated municipal wastewater.

The idea of using constructed wetlands for the treatment and improving of wastewater emerged in the second half of the last century. Despite relatively wide use of this environmentally friendly technology, relatively little is known about the microbial populations involved in biotransformation and removal of contaminants in this system. The aim of the current study was to investigate the assembly and function of microbial populations in vertical-flow constructed wetland microcosms designed to improve the quality of wastewater after activated sludge treatment. Also, the performance of 3-year-old wetland ponds was investigated. Even though the quality of the influent water was relatively high, improvement in water parameters such as coliform level, ammonia concentration, BOD, and TSS was observed. The performance of the wetland ponds was comparable to that of the microcosms. The microbial community composition of the biofilm formed on the surface of gravel particles in vegetated and plant-free microcosms was studied by denaturing gradient gel electrophoresis (DGGE) and sequencing of 16S rRNA gene fragments. Highly complex bacterial diversity was observed in the biofilm. Cluster analysis of DGGE patterns demonstrated that depth within the wetland microcosm has a stronger effect on microbial community composition of the biofilm formed on wetland matrix than vegetation. Measurements of fluorescein diacetate hydrolysis activity and nitrification potential revealed that hydrolytic activity was affected by both microcosm depth and vegetation presence, whereas nitrification potential was mostly influenced by depth. Resolving the bacterial assemblage of wetland biofilm, which often is considered a black box, will help to understand the interactions involved in the development of diverse and mature biofilm and its function.