Municipal wastewater treatment plant showing a potential reservoir for clinically relevant MDR bacterial strains co-occurrence of ESBL genes and integron-integrase genes.

Municipal wastewater treatment plants (MWWTPs) are a milieu for co-occurrence of multiple antibiotic resistance genes (ARGs). This facilitates mixing and genetic exchange; and promotes dissemination of multidrug resistance (MDR) to wastewater bacterial communities which is hazardous for the effluent receiving environment. This study investigated the co-occurrence of extended-spectrum beta-lactamase (ESBL) genes (blaTEM, blaCTX-M, blaSHV, blaOXA), and integron-integrase genes (intI1, intI2, intI3) in MDR bacteria isolated from the Bharwara MWWTP in Lucknow, India. Thirty-one MDR bacterial colonies resistant to three or more antibiotics were isolated from three treatment stages of this MWWTP. Six of these: Staphylococcus aureus, Serratia marcescens, Salmonella enterica, Shigella sonnei, Escherichia coli, and Bacillus sp. Had co-occurrence of ESBL and integron-integrase genes. These six isolates were examined for the occurrence of MDR efflux genes (qacA, acrB) and ARGs (aac(3)-1, qnrA1, tetA, vanA) and tested for resistance against 12 different antibiotics. The highest resistance was against penicillin-G (100%) and lowest for chloramphenicol (16.66%). Bacillus sp. Isolate BWKRC6 had the highest co-occurrence of antibiotic resistance-determining genes and was resistant to all the 12 antibiotics tested. The co-occurrence of ESBL, integron-integrase, antibiotic resistance-determining and MDR efflux genes in bacteria isolated from the Bharwara MWWTP indicates that the wastewaters of this treatment plant may have become a hotspot for MDR bacteria and may present human and environmental health hazards. Therefore, there is need for a rapid action to limit the spread of this threat. Public regulatory authorities must urgently implement measures to prevent MWWTPs becoming reservoirs for evolution of antibiotic resistance genes and development of antibiotic resistance.

Arbuscular mycorrhizas amplify the risk of heavy metal transfer to human food chain from fly ash ameliorated agricultural soils.

Soil contaminants threaten global food security by posing threats to food safety through food chain pollution. Fly ash is a potential agent of soil contamination that contains heavy metals and hazardous pollutants. However, being rich in macro- and micronutrients that have direct beneficial effects on plant growth, fly ash has been recommended as a low-cost soil ameliorant in agriculture in countries of the Global South. Arbuscular mycorrhizal fungi (AMF), ubiquitous in agricultural soils, enhance efficiency of plant nutrient uptake from soils but can equally increase uptake of toxic pollutants from fly ash ameliorated soils to edible crop tissues. We investigated AMF-mediated amplification of nutrient and heavy metal uptake from fly ash amended soils to shoots, roots and grains of barley. We used a microcosm-based experiment to analyse the impacts of fly ash amendments to soil in concentrations of 0 (control), 15, 30 or 50% respectively, on root colonization by AMF Rhizophagus irregularis and AMF-mediated transfer of N, P and heavy metals: Ni, Co, Pb and Cr to barley tissues. These concentrations of fly ash are equivalent to 0, 137, 275 and 458 t ha-1 respectively, in soil. Root AMF colonization correlated negatively with fly ash concentration and was not detected at 50% fly ash amendment. Shoots, roots and grains of mycorrhizal barley grown with 15, 30 and 50% fly ash amendments had significantly higher concentrations of Ni, Co, Pb and Cr compared to the control and their respective non-mycorrhizal counterparts. Presence of heavy metals in barley plants grown with fly ash amended soil and their increased AMF-mediated translocation to edible grains may significantly enhance the volume of heavy metals entering the human food chain. We recommend careful assessment of manipulation of agricultural soils with fly ash as heavy metal accumulation in agricultural soils and human tissues may cause irreversible damage.

Arbuscular mycorrhizas accelerate the degradation of colour containing organic pollutants present in distillery spent wash leachates.

Distillery spent wash (DSW) from molasses-based distilleries is being used as a low-cost alternative to chemical fertilizers in countries like India and Brazil. However, using DSW as a fertilizer substitute causes organic pollutant leaching, including melanoidins and caramel colourants that turn bodies of water dark brown. This study investigated the arbuscular mycorrhiza (AM) mediated degradation of organic pollutants in DSW. Mycorrhizal and non-mycorrhizal Sorghum bicolor were grown in microcosms for 16 weeks. The plants were fertilized with either raw DSW or Hoagland solution. Leachates draining from the microcosms after fertilization were collected three times in 30-day intervals. Each 30-day collection was preceded by two fertilizations. A gas chromatography-mass spectrometry comparative analyses of raw DSW with leachates of the third collection from mycorrhizal and non-mycorrhizal microcosms was made. Sixty-five and 42 complex organic compounds were detected in raw DSW and leachate collected from the non-mycorrhizal pots respectively. Only 26 compounds were detected in leachate collected from mycorrhizal pots. Absent from leachate of the mycorrhizal pots were: colour-containing organic compounds diacetone alcohol; 3-amino-2-cyano-6-methyl-6,7-dihydrothieno[2,3-b]pyrazine S-oxide; cyclohexane; 1,2-benzenedicarboxylic acid, butyl 8-methylnonyl ester; 2-pyrrolidinone; and acetic acid, dodecyl ester present in raw DSW. The results indicate that AM fungi can degrade organic pollutants in DSW.

Plant hosts may influence arbuscular mycorrhizal fungal community composition in mangrove estuaries.

We investigated the role of plant host and soil variables in determining arbuscular mycorrhizal fungi (AMF) community composition in plant roots of two spatially separated mangrove estuaries on the rivers Aghanashini (14° 30' 30″ N-74° 22' 44″ E) and Gangavali (14° 35' 26″ N-74° 17' 51″ E) on the west coast of India. Both mangrove estuaries had similar plant species composition but differed in soil chemistries.We amplified a 550-bp portion of 18S small subunit (SSU) rDNA from mangrove plant roots and analysed it by restriction fragment length polymorphism (RFLP). Clones representing unique RFLP patterns were sequenced. A total of 736 clones were obtained from roots of seven and five plant species sampled at Aghanashini and Gangavali, respectively. AMF phylotype numbers in plant roots at Aghanashini (12) were higher than at Gangavali (9) indicating quantitative differences in the AMF community composition in plant roots at the two mangrove estuaries. Because both estuaries had similar plant species composition, the quantitative difference in AMF communities between the estuaries could be an attribute of the differences in rhizospheric chemistry between the two sites.Non-metric multidimensional scaling (NMDS) revealed overlap in the AMF communities of the two sites. Three and two AMF phylotypes had significant indicator value indices with specific hosts at Aghanashini and Gangavali, respectively. Environmental vector fitting to NMDS ordination did not reveal a significant effect of any soil variable on AMF composition at the two sites. However, significant effects of both plant hosts and sites were observed on rhizospheric P. Our results indicate that root AMF community composition may be an outcome of plant response to rhizospheric variables. This suggests that plant identity may have a primary role in shaping AMF communities in mangroves.

Gharial nesting in a reservoir is limited by reduced river flow and by increased bank vegetation.

The gharial (Gavialis gangeticus Gmelin) is a fish-eating specialist crocodylian, endemic to south Asia, and critically endangered in its few remaining wild localities. A secondary gharial population resides in riverine-reservoir habitat adjacent to the Nepal border, within the Katerniaghat Wildlife Sanctuary (KWS), and nests along a 10 km riverbank of the Girwa River. A natural channel shift in the mainstream Karnali River (upstream in Nepal) has reduced seasonal flow in the Girwa stretch where gharials nest, coincident with a gradual loss of nest sites, which in turn was related to an overall shift to woody vegetation at these sites. To understand how these changes in riparian vegetation on riverbanks were related to gharial nesting, we sampled vegetation at these sites from 2017 to 2019, and derived an Enhanced Vegetation Index (EVI) from LANDSAT 8 satellite data to quantify riverside vegetation from 1988 through 2019. We found that sampled sites transitioned to woody cover, the number of nesting sites declined, and the number of nests were reduced by > 40%. At these sites, after the channel shift, woody vegetation replaced open sites that predominated prior to the channel shift. Our findings indicate that the lack of open riverbanks and the increase in woody vegetation at potential nesting sites threatens the reproductive success of the KWS gharial population. This population persists today in a regulated river ecosystem, and nests in an altered riparian habitat which appears to be increasingly unsuitable for the continued successful recruitment of breeding adults. This second-ranking, critically endangered remnant population may have incurred an "extinction debt" by living in a reservoir that will lead to its eventual extirpation.

HCN-producing Pseudomonas protegens CHA0 affects intraradical viability of Rhizophagus irregularis in Sorghum vulgare roots.

Arbuscular mycorrhizal fungi (AMF) and plant growth-promoting rhizobacteria inhabit the plant rhizosphere. Both functional groups can influence plant community structures, and interactions between them can vary from being synergistic to antagonistic. HCN-producing Pseudomonas protegens CHA0 is a plant growth-promoting rhizobacterium. P. protegens CHA0 has been shown to weakly attach to AMF hyphae. Here, we analyze the effect of P. protegens CHA0 on the viability of intraradical AMF hyphae. Using pot experiments, we have grown mycorrhizal and nonmycorrhizal Sorghum vulgare var. M35 with P. protegens CHA0 or HCN- mutant P. protegens CHA77, which did not produce HCN. Mycorrhizal and nonmycorrhizal Sorghum grown without CHA0 or CHA77 served as the control. While metabolically active AMF was not detected in mycorrhizal plants grown with HCN+ CHA0, the percentage of root colonization of metabolically active AMF in plants grown with HCN- CHA77 was lower than in the control. Root phosphorus was highest in mycorrhizal plants grown with HCN+ CHA0, but root Fe was higher in plants grown with the bacterial strains. Our results indicate that HCN-producing P. protegens can affect the viability of intraradical AMF.

Exposure to ionizing radiation affects the growth of ectomycorrhizal fungi and induces increased melanin production and increased capacities of reactive oxygen species scavenging enzymes.

Ectomycorrhizal (EM) fungi form symbioses with dominant tree families in boreal, temperate and tropical ecosystems and are important drivers of ecosystem function. EM fungal hyphae extend over a large area making them susceptible to enhanced radiation levels from naturally occurring or anthropogenically originating radioisotopes in the rhizosphere. In this study, the in-vitro effects of ionizing radiation on the growth and biomass of EM fungi Suillus luteus, S. bovinus and Rhizopogon luteolus were investigated. EM fungal cultures were exposed to gamma radiation from a 137Cs source for 137 h in darkness at 21 °C at dose rates of 404, 108.5 and 54.9 mGy h-1 resulting in total absorbed doses of 55.21, 14.82 and 7.50 Gy respectively. Cultures grown in the dark at 21 °C but not exposed to the 137Cs source served as the control. Our results show that EM fungi vary in their sensitivity to ionizing radiation. EM fungi used in this study produced melanin and reactive oxygen species scavenging enzymes such as catalase and superoxide dismutase as a response to ionizing radiation.

Symbiotic soil fungi enhance ecosystem resilience to climate change.

Substantial amounts of nutrients are lost from soils through leaching. These losses can be environmentally damaging, causing groundwater eutrophication and also comprise an economic burden in terms of lost agricultural production. More intense precipitation events caused by climate change will likely aggravate this problem. So far it is unresolved to which extent soil biota can make ecosystems more resilient to climate change and reduce nutrient leaching losses when rainfall intensity increases. In this study, we focused on arbuscular mycorrhizal (AM) fungi, common soil fungi that form symbiotic associations with most land plants and which increase plant nutrient uptake. We hypothesized that AM fungi mitigate nutrient losses following intensive precipitation events (higher amount of precipitation and rain events frequency). To test this, we manipulated the presence of AM fungi in model grassland communities subjected to two rainfall scenarios: moderate and high rainfall intensity. The total amount of nutrients lost through leaching increased substantially with higher rainfall intensity. The presence of AM fungi reduced phosphorus losses by 50% under both rainfall scenarios and nitrogen losses by 40% under high rainfall intensity. Thus, the presence of AM fungi enhanced the nutrient interception ability of soils, and AM fungi reduced the nutrient leaching risk when rainfall intensity increases. These findings are especially relevant in areas with high rainfall intensity (e.g., such as the tropics) and for ecosystems that will experience increased rainfall due to climate change. Overall, this work demonstrates that soil biota such as AM fungi can enhance ecosystem resilience and reduce the negative impact of increased precipitation on nutrient losses.

Rhizophagus irregularis MUCL 41833 can colonize and improve P uptake of Plantago lanceolata after exposure to ionizing gamma radiation in root organ culture.

Long-lived radionuclides such as (90)Sr and (137)Cs can be naturally or accidentally deposited in the upper soil layers where they emit ß/γ radiation. Previous studies have shown that arbuscular mycorrhizal fungi (AMF) can accumulate and transfer radionuclides from soil to plant, but there have been no studies on the direct impact of ionizing radiation on AMF. In this study, root organ cultures of the AMF Rhizophagus irregularis MUCL 41833 were exposed to 15.37, 30.35, and 113.03 Gy gamma radiation from a (137)Cs source. Exposed spores were subsequently inoculated to Plantago lanceolata seedlings in pots, and root colonization and P uptake evaluated. P. lanceolata seedlings inoculated with non-irradiated AMF spores or with spores irradiated with up to 30.35 Gy gamma radiation had similar levels of root colonization. Spores irradiated with 113.03 Gy gamma radiation failed to colonize P. lanceolata roots. P content of plants inoculated with non-irradiated spores or of plants inoculated with spores irradiated with up to 30.35 Gy gamma radiation was higher than in non-mycorrhizal plants or plants inoculated with spores irradiated with 113.03 Gy gamma radiation. These results demonstrate that spores of R. irregularis MUCL 41833 are tolerant to chronic ionizing radiation at high doses.

Soil moisture--a regulator of arbuscular mycorrhizal fungal community assembly and symbiotic phosphorus uptake.

Multiple species of arbuscular mycorrhizal fungi (AMF) can colonize roots of an individual plant species but factors which determine the selection of a particular AMF species in a plant root are largely unknown. The present work analysed the effects of drought, flooding and optimal soil moisture (15-20 %) on AMF community composition and structure in Sorghum vulgare roots, using PCR-RFLP. Rhizophagus irregularis (isolate BEG 21), and rhizosphere soil (mixed inoculum) of Heteropogon contortus, a perennial C4 grass, collected from the semi-arid Delhi ridge, were used as AMF inocula. Soil moisture functioned as an abiotic filter and affected AMF community assembly inside plant roots by regulating AMF colonization and phylotype diversity. Roots of plants in flooded soils had lowest AMF diversity whilst root AMF diversity was highest under the soil moisture regime of 15-20 %. Although plant biomass was not affected, root P uptake was significantly influenced by soil moisture. Plants colonized with R. irregularis or mixed AMF inoculum showed higher root P uptake than non-mycorrhizal plants in drought and control treatments. No differences in root P levels were found in the flooded treatment between plants colonized with R. irregularis and non-mycorrhizal plants, whilst under the same treatment, root P uptake was lower in plants colonized with mixed AMF inoculum than in non-mycorrhizal plants.

Pseudomonas fluorescens CHA0 can kill subterranean termite Odontotermes obesus by inhibiting cytochrome c oxidase of the termite respiratory chain.

Pseudomonas fluorescens CHA0 has been shown to suppress the growth of a wide range of microbial plant pathogens as well as invertebrate pests such as root nematodes. Hydrogen cyanide, a secondary metabolite produced by the bacterium, has been credited as being one of the determinants of its biocontrol ability. The use of biocontrol agents against social insect pests such as termite Odontotermes obesus has limitations because of behavioural adaptations that include (1) removal of the pathogen when grooming by the termites and (2) isolation of infested members of the colony. In this study, we show that cyanide of bacterial origin may inhibit cytochrome c oxidase (CCO) of the termite respiratory chain and demonstrate that HCN-producing bacteria such as P. fluorescens can actually kill a macroscopic insect pest by cyanide poisoning. This ability of pseudomonad metabolites such as cyanide, which can bring about pest death by blocking respiration through inhibition of CCO rather than infection or predation, can potentially overcome the behavioural adaptations of social insect pests such as termites and represents an attractive option for insect pest management.

Hydrogen cyanide-producing rhizobacteria kill subterranean termite Odontotermes obesus (Rambur) by cyanide poisoning under in vitro conditions.

The subterranean termite Odontotermes obesus is an important pest of the Indian subcontinent, causing extensive damage to major agricultural crops and forest plantation trees. Control of termites by strategies employing their parasites has limitations because they have evolved a complex social structure, immune responses, and adaptive behavior toward pathogen-infected individuals. Nonparasitic rhizobacteria that produce harmful metabolites might facilitate the biocontrol of termites. In the present investigation, three different species of hydrogen cyanide-producing rhizobacteria were tested for their potential to kill O. obesus. The three bacterial species were found to be effective in killing the termites under in vitro conditions.