Identification and Evaluation of Reference Genes for Quantitative PCR Normalization in Alligator Weed Flea Beetle (Coleoptera: Chrysomelidae)

Stably expressed reference genes are critical internal standards for the quantification of gene transcription levels using quantitative real-time PCR. Housekeeping genes are commonly used as reference genes but their expressions were variable depending on experimental conditions in many insect species studied. Here we report the identification and evaluation of 10 housekeeping genes in alligator weed flea beetle, Agasicles hygrophila Selman & Vogt (Coleoptera: Chrysomelidae), a biocontrol agent of alligator weed. The 10 housekeeping genes are: beta-actin (Actin), ribosomal protein L13A (PRL13a), succinate dehydrogenase complex subunit A (SDHA), ribosomal protein S20 (RPS20), ribosomal protein S13 (RPS13), glyceraldehyde phosphate dehydrogenase (GAPDH), TATA-box-binding protein (TBP), ribosomal protein L32 (RPL32), tubulin alpha-1 chain (TUBULIN), and elongation factor-1 alpha (ELF). Five programs, geNorm, NormFinder, BestKeeper, ΔCt method, and RefFinder, were used to evaluate the expression stability of the 10 genes among various A. hygrophila body parts and with different nutrient types (starvation, diet types). The expression stability analysis showed that RPS32 and RPL13a were reliable reference genes for the study of gene transcription in different body parts; Actin and RPL13a were optimal reference genes for different nutrient types. The selections of reference genes were validated using a CarE gene (GeneBank No: KX353552). The results of this study provide useful bases for studies of gene expression in various aspects relating to A. hygrophila.

Improving the productivity of okra (Abelmoschus esculentus L.) by strengthening the impact of applied nutrients through alligator weed compost

Weeds produce huge biomass by competing with the main crop for resources has been well established by numerous investigators. The use of weeds, especially aquatic weeds, as an organic source of nutrients such as compost is lacking in the literature. We hypothesized that the use of aquatic weeds i.e. alligator as compost would limit the use of inorganic fertilizers in okra and it would increase the quality of produce. To maximize the effect of inorganic nutrients, it would be easier to use the alligator weed compost as a natural source of nutrients. The crop was sown on 22 July, 2017 and 20 July, 2018 in a randomized complete block design having four replications. The net plot size was 1.8 m x 6 m. The alligator weed compost along with NPK was applied at the time of sowing. Alligator weed compost was applied along with different combinations of NPK i.e. control (recommended dose of NPK 159-114-93 kg ha-1 ), compost application of 250 kg ha-1 , compost application of 500 kg ha-1 , compost application of 250 kg ha-1 with 75% recommended NPK, compost application of 250 kg ha-1 with 50% recommended NPK, compost application of 250 kg ha-1 with 25% recommended NPK, compost application of 500 kg ha-1 compost with 75% recommended NPK, compost application of 500 kg ha-1 with 50% recommended NPK and compost application of 500 kg ha-1 with 25% recommended NPK. Compost application of 250 kg ha-1 with 50% recommended NPK significantly reduced the number of days to flowering and increased plant height (cm) at flowering and plant maturity over the alone NPK and other combinations of compost and NPK. Pod yield and yield components were outclassed by the compost application of 250 kg ha-1 with 50% recommended NPK over the other combinations. For example, number of pods per plant and pod yield (t ha-1 ) were increased by 26%, 20%, respectively averaged across during both years of study over the recommended dose of NPK alone. Similarly, application of compost at 500 kg ha-1 along with NPK 50% of recommended dose enhanced pod potassium and ascorbic acid contents by 76% and 35%, respectively averaged across during both years of study over the control dose of NPK. Conclusively, alligator weed organic source of nutrients could reduce NPK fertilizers as it holds the nutrients applied and also provides the essential nutrients. In future climates, alligator weed compost would be a sustainable and environment friendly approach.

Opposing effects of plant growth regulators via clonal integration on apical and basal performance in alligator weed

Aims Invasive plants are a major threat to biodiversity and may adversely affect food security. Clonal integration enables the sharing of resources between connected ramets and can enhance plant performance in many invasive species. However, few studies have examined the role of clonal integration when weeds are exposed to plant growth regulators (PGRs). PGRs are used extensively in agriculture and may affect nearby weeds through soil leaching, erosion, and runoff. Our aim was to investigate the effects of clonal integration on growth in a noxious weed, Alternanthera philoxeroides (alligator weed), in response to two PGRs frequently used in agriculture, gibberellins (GA) and paclobutrazol (PAC). Methods Ramets of A. philoxeroides were propagated in the greenhouse, and treated with PGRs. PGRs were applied to the older ramets (i.e., ‘basal’ part), with half of the plants having the stems between the apical (younger) and basal parts left connected, while the remaining plants had the stems between the two parts severed. Following the growing period, plants were measured for growth traits. Important Findings We found that GA and PAC had contrasting effects on plant growth. GA significantly promoted above-ground growth of the apical ramets via clonal integration. Alternatively, PAC inhibited above-ground growth in the basal and apical parts, and enhanced below-ground growth of the basal and apical ramets through clonal integration. Our results highlight how clonal integration can promote growth in A. philoxeroides following the application of PGRs, which is likely an important mechanism for this species to invade new environments.

Antibacterial activity of Alternanthera philoxeroides (Mart.) Griseb. against bacterial phytopathogens: Erwinia carotovora, Ralstonia solanacearum and Xanthomonas axonopodis

We investigated the antibacterial activity of alligator weed (Alternanthera philoxeroides) organic extracts against three bacterial phytopathogens (Erwinia carotovora, Ralstonia solanacearum and Xanthomonas axonopodis). The extracts were prepared by soaking the dry powder of leaf, stem and root of A. philoxeroides into methanol, n-hexane, chloroform and ethyl acetate. The disk diffusion method was used to determine the antibacterial activity at 100 mg/mL extract concentration. The n-hexane extract of A. philoxeroides leaves showed the maximum inhibition zone diameter (IZD)= 28.1 mm against R. solanacearum, while, the corresponding value for the positive control (Penicillin) was 48 mm IZD. There was no antibacterial activity of negative control, dimethyl sulfoxide (DMSO). Gas Chromatography Mass-Spectrometry (GC-MS) analysis revealed the presence of acetic acid, 2-(2-methoxycarbonylamino-5-nitrophenylthio)-, methyl ester, at the highest concentration (31.9 %), followed by 1, 4-benzenediol, 2, 5-bis (1,1-dimethylethyl)- (15.06 %),. It was concluded that the observed biological activity in this study may be due to the presence of these compounds.

Alternanthera philoxeroides (alligator weed).

A. philoxeroides is one of the worst weeds in the world because it invades both terrestrial and aquatic habitats. The aquatic form of the plant has the potential to become a serious threat to rivers, waterways, wetlands and irrigation systems. The terrestrial form grows forming dense mats with a massive underground rhizomatous root system (ISSG, 2016). This weed is extremely difficult to control, is able to reproduce from plant fragments and grows in a wide range of climates and habitats, including terrestrial areas. In aquatic habitats it has deleterious effects on other plants and animals, water quality, aesthetics, vector populations, water flow, flooding and sedimentation. In terrestrial situations, it degrades riverbanks, pastures, and agricultural lands producing massive underground lignified root systems penetrating up to 50-60 cm deep. Currently, A. philoxeroides is listed as invasive in the United States, Puerto Rico, France, Italy, India, Sri Lanka, China, Taiwan, Indonesia, Myanmar, Singapore, Australia and New Zealand (Weber et al., 2008; Chandra, 2012; Rojas-Sandoval and Acevedo-Rodriguez, 2015; DAISIE, 2016; USDA-ARS, 2016; USDA-NRCS, 2016; Weeds of Australia, 2016). Once established, it behaves as an aggressive invader with the capability to totally disrupt natural aquatic ecosystems, shoreline vegetation and terrestrial and semi-aquatic environments (ISSG, 2016; USDA-NRCS, 2016).

Composition Characteristics of Organic Matter and Bacterial Communities under the Alternanthera philoxeroide Invasion in Wetlands

The influence of Alternanthera philoxeroide (alligator weed) invasion on wetland organic matter (OM) accumulation and bacterial changes is rarely studied, but is possibly an important step for revealing the invasion mechanism. Thus, the distribution characteristics of light fraction organic carbon and nitrogen (LFOC and LFON), and heavy fractions organic carbon and nitrogen (HFOC and HFON) were analyzed. Sampling was done on two sediment depths (0–15 cm and 15–25 cm) of invaded and normal habitats of two natural wetlands and two constructed wetlands, and bacterial taxa and composition in surface sediments were also analyzed by high-throughput sequencing. In the surface sediments, the LFOC and LFON contents were significantly higher in the constructed wetlands (0.791 and 0.043 g·kg−1) than in the natural wetlands (0.500 and 0.022 g·kg−1), and the contents of the C and N fractions were also prominently higher in the invaded areas than in normal wetland habitats. The OM storage was relatively stable. Proteobacteria (55.94%), Bacteroidetes (5.74%), Acidobacteria (6.66%), and Chloroflexi (4.67%) were the dominant bacterial phyla in the wetlands. The abundance of Acidobacteria, Actinobacteria, and Gemmatimonadetes were significantly higher in the invaded areas than in the normal habitats. The relative high abundance-based coverage estimator (ACE) index in the constructed wetlands and invaded areas suggested the corresponding high bacterial diversity. The significant and positive relationship between Acidobacteria and organic nitrogen concentrations suggested their potential and positive interrelationships. This study demonstrated that the alligator weed invasion could significantly change the compositions of sediment organic matterand bacteria, thus further changing the nutrition cycle and wetland microhabitat.

Induction of Heat Shock Protein Genes is the Hallmark of Egg Heat Tolerance in Agasicles hygrophila (Coleoptera: Chrysomelidae)

Insects are ecotothermic organisms. Their development, survival, reproduction as well as distribution and abundance are affected by temperature. Heat shock protein (HSP) gene expression is closely associated with temperature variation and influences the adaptation of organisms to adverse environments. The beetle Agasicles hygrophila has successfully been used for biological control of the invasive plant alligator weed (Alternanthera philoxeroides). As A. hygrophila populations are substantially inhibited by high temperatures in the summer, increasing global temperatures may limit the efficacy of this control agent. We previously established that A. hygrophila eggs have low tolerance to heat and this factored into the decreased numbers of A. hygrophila beetles at temperatures of 37.5°C and above. Here, we identified 26 HSP genes in A. hygrophila and examined the relationship between the transcript levels of these genes and heat tolerance. The temperature at which the expression of these 21 HSP genes peaked (Tpeak) was 37.5°C, which is in line with the limit of the high temperatures that A. hygrophila eggs tolerate. Therefore, we speculate that the Tpeak of HSP gene expression in eggs indicates the upper limit of temperatures that A. hygrophila eggs tolerate. This study identifies HSP genes as potential robust biomarkers and emphasizes that determining species’ heat tolerance in their natural habitats remains an important consideration for biocontrol. HSP gene expression data provide information about a species’ heat tolerance and may be used to predict its geographical distribution.