Characterisation of root amino acid exudation in white clover (Trifolium repens L.)

Lateral roots are crucial for the plasticity of root responses to environmental conditions in soil. The bacterivorous microfauna has been shown to increase root branching and to foster auxin producing soil bacteria. However, information on modifications of plant internal auxin content by soil bacteria and bacterivores is missing. Therefore, the effects of a rhizosphere bacterial community and a common soil amoeba (Acanthamoeba castellanii) on root branching and on auxin (indole-3-acetic acid) metabolism in Lepidium sativum and Arabidopsis thaliana were investigated. In a first experimental series, bacteria increased conjugated auxin concentrations in L. sativum shoots, but did not alter free bioactive auxin content nor root branching. In contrast, in presence of soil bacteria plus amoebae free auxin concentrations in shoots and root branching increased, demonstrating that effects of bacteria on auxin metabolism in plants were strongly modified by the bacterivorous amoebae. In a second experiment, A. thaliana reporter plants for auxin (DR5) and cytokinin (ARR5) responded similarly with increased root branching in the presence of amoebae. Surprisingly, in reporter plants cytokinin but not auxin responses were detectable, accompanied by higher soil nitrate concentrations in the presence of amoebae. Likely, increased nitrate concentrations in the rhizosphere led to an accumulation of cytokinin and interactions with free auxin in plants and finally to increased root growth in the presence of amoebae. Altogether, the results show that mutual control mechanisms exist between plant hormone metabolism and microbial signalling, and that effects on hormonal concentrations of plants by free-living bacteria are strongly influenced by bacterial grazers like amoebae.     

Influence of arbuscular mycorrhizal fungi on microbes and enzymes of soils from different cultivated densities of red clover

Effects of arbuscular mycorrhizal fungus (AMF)Glomus mosseae on plant growth, soil microbial populations and enzymes activities of soils were studied in red clover (Trifolium pratense L.) grown in pots at different cultivated densities. Seeds of red clover were sown with 50 g inoculums ofG. mosseae per pot. After a week, the plants were thinned to 20, 30, 40, 50 and 60 seedlings per pot. Three months after treatment, AMF inoculation significantly stimulated plant growth. Quantities of vesicles and spores, arbuscules and hyphae were the highest when 30 and 50 seedlings were grown per pot, respectively. However, no root was infected in control plants. In all the soil sites, the numbers of fungi and bacteria were followed in the order: root > root surface > rhizospheric. It was indicated that arbuscular mycorrhizal fungus decreased the numbers of fungi and bacteria but improved growth of actinomycetes. Compared to control plants, AMF stimulated activities of phosphatase and urease but decreased invertase.     

Isolation of endophytic bacteria from embryogenic suspension culture of banana and assessment of their plant growth promoting properties

Endophytic microorganisms have been reported from various plants. In the current study, somatic embryogenic cultures of banana (Musa accuminata AAA cv. Grand Naine) were found to have association with endophytic bacteria which were present initially in a covert state. The presence of bacteria was detected only in suspension cultures derived from the somatic embryogenic cultures. The bacteria isolated from embryogenic cell suspension culture were identified as Ralstonia sp. and Bacillus sp. The Ralstonia sp. interestingly showed the presence of various plant growth promoting properties including indole acetic acid and siderophore production. Also the strain was found to have the potential to solubilize phosphate and produce ammonia. Growth enhancement effect of Ralstonia sp. on Vigna radiata seedlings showed promising results and the growth parameters were found to be statistically significant when compared to control. Identification and confirmation of the plant growth promoting properties of Ralstonia sp. makes the study significant with promising applications.     

Protozoa Drive the Dynamics of Culturable Biocontrol Bacterial Communities

Some soil bacteria protect plants against soil-borne diseases by producing toxic secondary metabolites. Such beneficial biocontrol bacteria can be used in agricultural systems as alternative to agrochemicals. The broad spectrum toxins responsible for plant protection also inhibit predation by protozoa and nematodes, the main consumers of bacteria in soil. Therefore, predation pressure may favour biocontrol bacteria and contribute to plant health. We analyzed the effect of Acanthamoeba castellanii on semi-natural soil bacterial communities in a microcosm experiment. We determined the frequency of culturable bacteria carrying genes responsible for the production of the antifungal compounds 2,4-diacetylphloroglucinol (DAPG), pyrrolnitrin (PRN) and hydrogen cyanide (HCN) in presence and absence of A. castellanii. We then measured if amoebae affected soil suppressiveness in a bioassay with sugar beet seedlings confronted to the fungal pathogen Rhizoctonia solani. Amoebae increased the frequency of both DAPG and HCN positive bacteria in later plant growth phases (2 and 3 weeks), as well as the average number of biocontrol genes per bacterium. The abundance of DAPG positive bacteria correlated with disease suppression, suggesting that their promotion by amoebae may enhance soil health. However, the net effect of amoebae on soil suppressiveness was neutral to slightly negative, possibly because amoebae slow down the establishment of biocontrol bacteria on the recently emerged seedlings used in the assay. The results indicate that microfaunal predators foster biocontrol bacterial communities. Understanding interactions between biocontrol bacteria and their predators may thus help developing environmentally friendly management practices of agricultural systems.     

Effect of <Emphasis Type="Italic">Pseudomonas</Emphasis> Bacteria on Peroxidase Activity in Wheat Plants when Infected with <Emphasis Type="Italic">Bipolaris sorokiniana</Emphasis>

We investigated the effect of treating soft wheat seeds (Triticum aestivum L.) with two Pseudomonas bacteria strains, isolated from earthworm coprolites, showing a significant antifungal and growth-promoting action in preliminary screening on the activity of guaiacol-dependant peroxidase under phytopathogenic load in the presence of Bipolaris sorokiniana (Sacc.) Shoemaker as a mechanism for inducing plant resistance to the pathogen. We established a statistically significant decrease (P < 0.05) in root rot disease incidence and severity during bacterization, which is indicative both of antifungal activity of the used bacterial isolates and of their successful colonizing the rhizosphere of wheat plants. We noted a response of free and weakly bound peroxidase of wheat plants to infection with B. sorokiniana: the enzyme activity increased during pathogenesis. Bacterization also increased peroxidase activity in plant leaves and roots, the greatest differences from non-bacterized plants being observed in wheat roots in the presence of the pathogen. We detected a direct link between peroxidase activity in wheat roots and leaf tissues in the absence of the pathogen and the feedback between peroxidase activity and plant infestation by the root rot pathogen. In the presence of the phytopathogen, there is a lack of correlation between peroxidase activity in wheat roots and leaves, and there is a shift of activity towards its increase in roots, which plays an important role in the development of systemic resistance against the root rot pathogen that penetrates into plants through the roots and root collar.     

Plant Growth Substances Produced by Azospirillum brasilense and Their Effect on the Growth of Pearl Millet (Pennisetum americanum L.)

Azospirillum brasilense, a nitrogen-fixing bacterium found in the rhizosphere of various grass species, was investigated to establish the effect on plant growth of growth substances produced by the bacteria. Thin-layer chromatography, high-pressure liquid chromatography, and bioassay were used to separate and identify plant growth substances produced by the bacteria in liquid culture. Indole acetic acid and indole lactic acid were produced by A. brasilense from tryptophan. Indole acetic acid production increased with increasing tryptophan concentration from 1 to 100 μg/ml. Indole acetic acid concentration also increased with the age of the culture until bacteria reached the stationary phase. Shaking favored the production of indole acetic acid, especially in a medium containing nitrogen. A small but biologically significant amount of gibberellin was detected in the culture medium. Also at least three cytokinin-like substances, equivalent to about 0.001 μg of kinetin per ml, were present. The morphology of pearl millet roots changed when plants in solution culture were inoculated. The number of lateral roots was increased, and all lateral roots were densely covered with root hairs. Experiments with pure plant hormones showed that gibberellin causes increased production of lateral roots. Cytokinin stimulated root hair formation, but reduced lateral root production and elongation of the main root. Combinations of indole acetic acid, gibberellin, and kinetin produced changes in root morphology of pearl millet similar to those produced by inoculation with A. brasilense.     

Productivity of soybean-rhizobium symbiosis after modification of root nodule bacteria activity with exogenous proteins

Plant growth experiments were conducted to assess symbiotic efficiency, photosynthetic rates, and the development of soybean (Glycine max (L.) Merrill) seedlings after seed inoculation with active and inactive strains of root nodule bacteria Bradyrhizobium japonicum preincubated in the presence homologous and heterologous proteins. The properties of active and inactive symbiotic strains were differentially modulated by homologous soybean lectin, which had a marked influence on plant physiological condition. The incubation of active rhizobia with a homologous lectin, i.e., lectin of the respective plant, increased the nitrogen-fixing activity of nodules and, consequently, elevated photosynthetic rates and weight increments in soybean plants. At the same time, the homologous lectin suppressed the symbiotic properties of inactive strain of nodule bacteria. The preincubation of rhizobia with a heterologous pea lectin had virtually no effect on functioning of symbiotic apparatus and photosynthetic rate, whereas the preincubation of root nodule bacteria with human albumin exerted an effect similar to that induced by a homologous lectin on symbiotic productivity.     

Acid phosphatase and leucine aminopeptidase isozymes as biochemical markers of homogeneity in oil seed rape androgenetic lines

Extracts of cotyledons of Brassica napus plants (seed progenies of doubled haploid plants) were separated by electrophoresis on polyacrylamide gels and stained for acid phosphatase (ACP-E.C. 3.1.3.2.) and leucine aminopeptidase (LAP-E.C. 3.4.11.1.) enzymes to investigate the possibility of utilising isozymes as markers of homogeneity (purity) of plant populations. One zone of activity for acid phosphatase and two zones of activity for leucine aminopeptidase were identified on gels, some variation in isozyme patterns occurred in several androgenetic lines. This method is appropriate and consistent for testing the homogeneity of breeding lines-progenies of double haploid (D.H.) plants.     

Effects of Interactions of Auxin-Producing Bacteria and Bacterial-Feeding Nematodes on Regulation of Peanut Growths

The influences of an IAA (indole-3-acetic acid)-producing bacterium (Bacillus megaterium) and two bacterial-feeding nematodes (Cephalobus sp. or Mesorhabditis sp.) on the growth of peanut (Arachis hypogaea L. cv. Haihua 1) after various durations of time were investigated in natural soils. The addition of bacteria and nematodes and incubation time all significantly affected plant growth, plant root growth, plant nutrient concentrations, soil nutrient concentrations, soil microorganisms and soil auxin concentration. The addition of nematodes caused greater increases in these indices than those of bacteria, while the addition of the combination of bacteria and nematodes caused further increases. After 42-day growth, the increases in soil respiration differed between the additions of two kinds of nematodes because of differences in their life strategies. The effects of the bacteria and nematodes on the nutrient and hormone concentrations were responsible for the increases in plant growth. These results indicate the potential for promoting plant growth via the addition of nematodes and bacteria to soil.     

Role of heavy metal resistant Ochrobactrum sp. and Bacillus spp. strains in bioremediation of a rice cultivar and their PGPR like activities

The present study demonstrates the metal toxicity ameliorating and growth promoting abilities of three different bacterial isolates when applied to rice as host plant. The three bacterial strains included a cadmium resistant Ochrobactrum sp., a lead resistant Bacillus sp. and an arsenic resistant Bacillus sp. designated as CdSP9, PbSP6, and AsSP9, respectively. When these isolates were used as inocula applied to metal-treated rice plants of variety Satabdi, the germination percentage, relative root elongation (RRE), amylase and protease activities were increased. The toxic effect of metal was reduced in presence of these bacteria. The overall biomass and root/shoot ratio were also enhanced by bacterial inoculation. Hydroponic studies showed that the superoxide dismutase (SOD) activity and malondialdehyde (MDA) level, which had been increased in the presence of metal stress in rice roots, were lowered by the bacterial inoculation. In addition, all three strains were 1-aminocyclopropane-1-carboxylate (ACC) deaminase and catalase positive, whereas siderophore producing ability was lacking in PbSP6. However, both PbSP6 and AsSP9 were protease positive and could hydrolyse starch. The data indicate that these bacteria have promise for bioremediation as well as for plant growth promotion.     

Pyruvate kinase, a possible regulatory enzyme in higher plants

A number of plant species were examined for the presence of pyruvate kinase (pyruvate-ATP phosphotransferase, EC 2.7.1.40), and of a phosphatase activity which hydrolyzes phosphoenolpyruvate. Of those examined, only cotton (Gossypium sp. L.) seeds were found to be sufficiently free of the phosphatase to permit a kinetic study of pyruvate kinase.     

Localization of acid phosphatase activity in maize root under phosphorus deficiency

The effect of phosphorus deficiency on acid phosphatase activity in the apical, middle and basal parts of the root of maize plants was followed. The supernatant obtained by centrifuging the homogenate of plant tissue at 1500 ×g was further centrifuged at 18 000 ×g, the sediment being marked as fraction II and the supernatant as fraction III. The results obtained document the fact that acid phosphatase activity of the two fractions of all analyzed root segments was higher in plants cultured in nutrient medium without phosphate than in those containing phosphorus in nutrient medium. In most cases this difference was significant to highly significant. The results of experiments proved unambiguously a higher enzymatic activity in all root segments in fraction III than in fraction II. In fraction III the highest acid phosphatase activity was found in the apical part, in fraction II in the basal part of the root.     

Symbiotic interactions between arbuscular mycorrhizal (AM) fungi and male papaya plants: Its status,role and implications

Experiments were conducted to study the arbuscular mycorrhizal (AM) status and its role in P-uptake through assay of root phosphatases activities in four varieties of male Carica papaya L. viz. CO-1, CO-2, Honey Dew and Washington during flowering stages. In the present study, mean total root colonization of AM fungi recorded peak increase in flowering stage-II while mean root phosphatase (acid and alkaline) activities recorded peak increase in flowering stage-I. Unlike root colonization and root phosphatase activities, spore density did not exhibit any definite patterns and recorded a narrow range of fluctuation during different flowering stages of male C. papaya. The study brought out the fact that root colonization and spore density of AM fungi along with root phosphatase activities varied significantly within the four varieties of male C. papaya plants during each flowering stage. The study also recorded consistently higher acid root phosphatase activity than alkaline root phosphatase activity under P-deficient, acidic soil conditions during all flowering stages of male C. papaya plants. Studies revealed that the root colonization of AM fungi influenced root phosphatase activities (acid and alkaline) positively and significantly during all flowering stages of male C. papaya plants. A total of twelve species of AM fungi belonging to five genera viz. Acaulospora, Dentiscutata, Gigaspora, Glomus, and Racocetra were recovered from the rhizosphere of male C. papaya plants.     

Morphological, physiological, cytological and phytochemical studies in diploid and colchicine-induced tetraploid plants of Echinacea purpurea (L.)

Echinacea purpurea (L.) is one of the important medicinal plant species. To obtain the tetraploid plants of Echinacea purpurea with improved medicinal qualities, the root tips of two true leaves seedlings were imbibed in 0.25 % (w/v) colchicine solution for 24, 48, 72, 96 and 168 h. The ploidy level of plants was determined by chromosome counting of root tip cells, and confirmed by flow cytometric analysis. Tetraploid induction occurred in seedlings treated for 24, 48 and 72 h at colchicine solution. The morphological, physiological, cytological, and phytochemical characteristics of diploid and colchicine-induced tetraploid plants were compared. Results indicated that tetraploid plants had considerable larger stomata, pollen grain, seed and flower. Moreover, chloroplast number in guard cells, amount of chlorophyll (a, b, and a + b), carotenoids as well as width and thickness of leaves were increased in tetraploids. However, stomata frequency, leaf index, plant height, and quantum efficiency of photosystem II in tetraploid were lower than diploid plants. High-performance liquid chromatography analysis showed that leaves of the tetraploid plants had more cichoric acid (45 %) and chlorogenic acid (71 %) than diploid plants. It was concluded that morphological and physiological characteristics can be used as useful parameters for preliminary screening of putative tetraploids in this species.     

Effects of nematodes,fungi and bacteria on the growth of young apple trees grown in apple replant disease soil

Growth, dry root weight of seedlings and root score of apple seedlings cv. McIntosh were reduced when soils were inoculated with Pratylenchus penetrans, Penicillium janthinellum, Constantinella terrestris, Trichoderma sp., and 4 strains of Bacillus subtilis. Trichoderma sp., and B-1 and B-26 strains of B. subtilis alone reduced plant growth but the combination of Trichoderma sp. + B. subtilis (B-1) and Trichoderma sp. + B. subtilis (B-26) increased plant height. Plant height, root weight and root score were significantly reduced when P. penetrans plus B. subtilis or P. penetrans plus fungi plus bacteria were present in the soil. It is suggested that fungi, bacteria, nematodes alone or their combinations such as nematodes plus bacteria or nematodes plus fungi plus bacteria may contribute towards the occurrence of apple replant disease.Contribution number 700.Contribution number 700.     

Differences in Endogenous Levels of Gibberellin-Like Substances in Nodules of Phaseolus lunatus L. Plants Inoculated with Two Rhizobium Strains

Lima bean plants (Phaseolus lunatus L.) inoculated with Rhizobium sp. strain 127E14, which lacks constitutive nitrate reductase activity, were significantly taller after 4 weeks of age than plants inoculated with strain 127E15, which contains constitutive nitrate reductase activity. Plants inoculated with either strain responded to application of 5 micrograms gibberellic acid per plant with rapid internode elongation; plants inoculated with strain 127E15 became less responsive to gibberellic acid from 3 to 5 weeks of age, while plants inoculated with strain 127E14 did not. The height of plants inoculated with strain 127E14 was reduced by 20% with application of gibberellin biosynthesis inhibitors to the roots, while height of plants inoculated with strain 127E15 was unaffected.     

The effect of plant growth-promoting rhizobacteria on the growth of rice (Oryza sativa L.) cropped in southern Brazilian fields

Background and Aims

Several strains of rhizobacteria may be found in the rhizospheric soil, on the root surface or in association with rice plants. These bacteria are able to colonize plant root systems and promote plant growth and crop yield through a variety of mechanisms. The objectives of this study were to isolate, identify, and characterize putative plant growth-promoting rhizobacteria (PGPR) associated with rice cropped in different areas of southern Brazil.

Methods

Bacterial strains were selectively isolated based on their growth on three selective semi-solid nitrogen-free media. Bacteria were identified at the genus level by PCR-RFLP 16S rRNA gene analysis and partial sequencing methodologies. Bacterial isolates were evaluated for their ability to produce indolic compounds and siderophores and to solubilize phosphate. In vitro biological nitrogen fixation and the ability to produce 1-aminocyclopropane-1-carboxylate deaminase were evaluated for each bacterial isolate used in the inoculation experiments.

Results

In total, 336 bacterial strains were isolated representing 31 different bacterial genera. Strains belonging to the genera Agrobacterium, Burkholderia, Enterobacter, and Pseudomonas were the most prominent isolates. Siderophore and indolic compounds producers were widely found among isolates, but 101 isolates were able to solubilize phosphate. Under gnotobiotic conditions, eight isolates were able to stimulate the growth of rice plants. Five of these eight isolates were also field tested in rice plants subjected to different nitrogen fertilization rates.

Conclusions

The results showed that the condition of half-fertilization plus separate inoculation with the isolates AC32 (Herbaspirillum sp.), AG15 (Burkholderia sp.), CA21 (Pseudacidovorax sp.), and UR51 (Azospirillum sp.) achieved rice growth similar to those achieved by full-fertilization without inoculation, thus highlighting the potential of these strains for formulating new bioinoculants for rice crops.     

Interactions between Zeldia Punctata (Cephalobidae) and bacteria in the presence or absence of maize plants

Bacterial-feeding nematodes constitute one of the primary grazers of soil bacteria. We investigated the effects of selective grazing of a representative nematode (Zeldia punctata, Cephalobidae) on nematode life history and population biology and on the soil microbial community. Firstly, we measured (i) the effect of five different bacterial strains on the nematode life cycle using petri dishes and (ii) the impact of bacterial inoculation on nematode population growth in a soil microcosm. Selection of the five bacterial strains was based on morphology, cell-wall characteristics and mucus production. Z. punctata development was strongly affected by the type of bacteria ingested, independent of experimental design. Bacterial cell-wall characteristics seemed to directly affect Z. punctata development since high nematode densities were only reached with gram-negative strains (Pseudomonas monteilii and Methylobacterium nodulans). In petri dishes, the filamentous organisms (Actinomyces sp.) and mucus-producing bacteria (Bradyrhizobium sp.) led to the least reproduction. Duration of the various nematode life phases (egg, juvenile, reproductive stage and non-reproductive stage) was significantly affected by the bacterial food source. Total life span varied from 12.5 days (Bradyrhizobium sp.) to 40 days (Pseudomonas monteilii). Secondly, we monitored the influence of Z. punctata on the indigenous soil microbial community in the presence or absence of a maize plantlet. Nematode inoculation led to an increase in bacterial activity (as measured by alkaline phosphatase activity) but did not significantly influence bacterial biomass. The genetic fingerprint (DGGE) of soil bacteria was more influenced by plant presence than by nematode inoculation. Nematode activity has important repercussions on N flux in the soil since inoculation of Z. punctata in the absence of plants resulted in a net increase of N mineralization (2 mg N per pot) while a decrease of mineral N (0.5 mg N per pot) was observed in the absence of the nematodes, due to bacterial immobilization. This study underscores the close relationship between selective bacterial grazing and nematode development. Nevertheless, the impact of nematode grazing on the overall soil microbial community seems to primarily affect microbial activity and relative dominance rather than microbial diversity.     

Molecular characterization and identification of plant growth promoting endophytic bacteria isolated from the root nodules of pea (Pisum sativum L.)

Root nodule accommodates various non-nodulating bacteria at varying densities. Present study was planned to identify and characterize the non-nodulating bacteria from the pea plant. Ten fast growing bacteria were isolated from the root nodules of cultivated pea plants. These bacterial isolates were unable to nodulate pea plants in nodulation assay, which indicate the non-rhizobial nature of these bacteria. Bacterial isolates were tested in vitro for plant growth promoting properties including indole acetic acid (IAA) production, nitrogen fixation, phosphate solubilization, root colonization and biofilm formation. Six isolates were able to produce IAA at varying level from 0.86 to 16.16 μg ml^?1, with the isolate MSP9 being most efficient. Only two isolates, MSP2 and MSP10, were able to fix nitrogen. All isolates were able to solubilize inorganic phosphorus ranging from 5.57 to 11.73 μg ml^?1, except MSP4. Bacterial isolates showed considerably better potential for colonization on pea roots. Isolates MSP9 and MSP10 were most efficient in biofilm formation on polyvinyl chloride, which indicated their potential to withstand various biotic and abiotic stresses, whereas the remaining isolates showed a very poor biofilm formation ability. The most efficient plant growth promoting agents, MSP9 and MSP10, were phylogenetically identified by 16S rRNA gene sequence analysis as Ochrobactrum and Enterobacter, respectively, with 99 % similarity. It is suggested the potential endophytic bacterial strains, Ochrobactrum sp. MSP9 and Enterobacter sp. MSP10, can be used as biofertilizers for various legume and non-legume crops after studying their interaction with the host crop and field evaluation.     

Differential Activity of Autochthonous Bacteria in Controlling Drought Stress in Native Lavandula and Salvia Plants Species Under Drought Conditions in Natural Arid Soil

The effectiveness of autochthonous plant growth-promoting rhizobacteria was studied in Lavandula dentata and Salvia officinalis growing in a natural arid Mediterranean soil under drought conditions. These bacteria identified as Bacillus megaterium (Bm), Enterobacter sp. (E), Bacillus thuringiensis (Bt), and Bacillus sp. (Bsp). Each bacteria has different potential to meliorate water limitation and alleviating drought stress in these two plant species. B. thuringiensis promoted growth and drought avoidance in Lavandula by increasing K content, by depressing stomatal conductance, and it controlled shoot proline accumulation. This bacterial effect on increasing drought tolerance was related to the decrease of glutathione reductase (GR) and ascorbate peroxidase (APX) that resulted sensitive indexes of lower cellular oxidative damage involved in the adaptative drought response in B. thuringiensis-inoculated Lavandula plants. In contrast, in Salvia, having intrinsic lower shoot/root ratio, higher stomatal conductance and lower APX and GR activities than Lavandula, the bacterial effects on nutritional, physiological and antioxidant enzymatic systems were lower. The benefit of bacteria depended on intrinsic stress tolerance of plant involved. Lavadula demonstrated a greater benefit than Salvia to control drought stress when inoculated with B. thuringiensis. The bacterial drought tolerance assessed as survival, proline, and indolacetic acid production showed the potential of this bacteria to help plants to grow under drought conditions. B. thuringiensis may be used for Lavandula plant establishment in arid environments. Particular characteristic of the plant species as low shoot/root ratio and high stomatal conductance are important factors controlling the bacterial effectiveness improving nutritional, physiological, and metabolic plant activities.