Optimum Growth Temperature Research Articles

Thermal adaptation of soil microbial growth traits in response to chronic warming.

Soils are the largest terrestrial carbon sink and the foundation of our food, fiber, and fuel systems. Healthy soils are carbon sinks, storing more carbon than they release. This reduces the amount of carbon dioxide released into the atmosphere and buffers against climate change. Soil microbes drive biogeochemical cycling and contribute to soil health through organic matter breakdown, plant growth promotion, and nutrient distribution. In this study, we determined how soil microbial growth traits respond to long-term soil warming. We found that bacterial isolates from warmed plots showed evidence of adaptation of optimum growth temperature. This suggests that increased microbial biomass and growth in a warming world could result in greater carbon storage. As temperatures increase, greater microbial activity may help reduce the soil carbon feedback loop. Our results provide insight on how atmospheric carbon cycling and soil health may respond in a warming world.

Larger diatoms are more sensitive to temperature changes and prone to succumb to warming stress

AbstractThe effects of ocean warming on phytoplankton have been extensively studied, but little has been documented on responses of differently sized diatoms to changes of temperature over wide ranges. We grew three diatom species, Thalassiosira pseudonana, Thalassiosira weissflogii, and Thalassiosira punctigera, with cell volumes of 199, 1805, and 13,599 μm3, respectively, at seven temperatures (5, 10, 15, 20, 25, 30, and 35°C) over 15 generations, and investigated their physiological responses. The optimum growth temperature (Topt), maximum growth rates (μmax), and thermal niche width (w) decreased by 16%, 48%, and 29% per order of magnitude in cell size increment, respectively. The cell volume of each species diminished significantly and linearly with increased temperatures, by 22% in T. punctigera, 17% in T. weissflogii, and 11% in T. pseudonana, respectively. However, the cellular biogenic silica (BSi) contents increased with rising temperature till the optimum growth temperature for growth, with the sensitivity of BSi to temperature changes (Q10BSi) ranging from 1.4 in the largest to 3.4 in the smallest diatom. The largest diatom T. punctigera showed the highest deactivation energy and succumbed to warmer conditions over its Topt, while the smallest was the most tolerant of warming. Although the moderate increase in growth temperature increased the diatoms' BSi levels, warming to temperatures higher than the optimum temperature for growth significantly reduced it. Our results suggest that diatoms acclimated to warmer conditions diminish their cell size but increase their mineralization of silica, which has profound significance in ocean carbon and silica biogeochemical cyclings.

Thermophilic Fungi as the Microbial Agents of Choice for the Industrial Co-Fermentation of Wood Wastes and Nitrogen-Rich Organic Wastes to Bio-Methane.

The novel industrial approach of co-fermenting wood wastes with agricultural wastes that are rich in nitrogen such as animal manures to produce bio-methane (renewable natural gas) fuel via thermophilic anaerobic digestion mimics an analogous process occurring in lower termites, but it relies instead on thermophilic fungi along with other thermophilic microorganisms comprising suitable bacteria and archaea. Wood microbial hydrolysis under thermophilic temperatures (range of 55 °C to 70 °C) and aerobic or micro-aerobic conditions constitutes the first step of the two-step (hydrolysis and fermentation) dry thermophilic anaerobic digestion industrial process, designated as "W2M3+2", that relies on thermophilic fungi species, most of which grow naturally in wood piles. Eleven thermophilic fungi have been identified as likely agents of the industrial process, and their known growth habitats and conditions have been reviewed. Future research is proposed such that the optimal growth temperature of these thermophilic fungi could be increased to the higher thermophilic range approaching 70 °C, and a tolerance to partial anaerobic conditions can be obtained by modifying the fungal microbiome via a symbiotic existence with bacteria and/or viruses.

Arthrobacter vasquezii sp. nov., isolated from a soil sample from Union Glacier, Antarctica.

A Gram-stain-positive, catalase-positive, non-motile bacteria, with a rod-coccus cycle (designated as EH-1B-1T) was isolated from a soil sample from Union Glacier in Ellsworth Mountains, Antarctica. Strain EH-1B-1T had an optimal growth temperature of 28 °C and grew at pH 7-10. The major cellular fatty acids were anteiso-C15 : 0, iso-C15 : 0, C16 : 0 and anteiso-C17 : 0. The G+C content based on the whole genome sequence was 63.1 mol%. Strain EH-1B-1T was most closely related to members of the genus Arthrobacter, namely Arthrobacter subterraneus and Arthrobacter tumbae. The strain grew on tryptic soy agar, Reasoner's 2A agar, lysogeny broth agar and nutrient agar. The average nucleotide identity and digital DNA-DNA hybridization values between strain EH-1B-1T and its closest reference type strains ranged from 78 to 88 % and from 20.9 to 36.3 %, respectively. Based on phenotypic, chemotypic and genotypic evidence, it is proposed that strain EH-1B-1T represents a novel species of Arthrobacter, for which the name Arthrobacter vasquezii sp. nov. is proposed, with strain EH-1B-1T (RGM 3386T=LMG 32961T) as the type strain.

Dispersal and Distribution of Thermophilic Endospores in Deep-Sea Ecosystems

The distance-decay relationship is a central concept in biogeography and spatial biodiversity, describing how two distinct entities decrease in similarity as the distance between them increases. The decay of community similarity with geographical distance is driven by multiple factors, such as gene drift, environmental selection, and the accumulation of mutations over time. While the distance-decay relationship has been recognized for several decades, there are certain circumstances where the biogeographical patterns of certain species and communities cannot be predicted by this relationship. The example addressed in this project is the case of thermophilic endospore-forming bacteria found in permanently cold deep ocean sediments. Thermophilic endospores (thermospores) are routinely found on the deep ocean floor, a permanently cold environment that does not support their metabolic activity (Hubert et al. 2009). Thermospores are metabolically dormant states developed by some thermophilic bacteria having optimal growth temperatures between 40°C and 70°C. There is evidence that these heat-loving bacteria originate from the deep subsurface and are transported upward to the deep ocean via geological features of the oceanic crust including geofluid fluxes in high-temperature axial systems and natural hydrocarbon seeps (Gittins et al. 2022). Due to the ability of endospores to stay viable for thousands of years and resist a wide range of physicochemical stressors, they can disperse over long distances while remaining unaffected by changing factors such as selection, drift, or mutation (Gittins et al. 2022; Fig. 1). Consequently, these thermospores have the potential to challenge the distance-decay relationship and exhibit unique biogeographical patterns. This work quantifies endospores in sediment cores at and around hydrocarbon seeps using cores from deep-sea Scotian Slope sediments. Given the challenge of quantifying specific groups of endospores (e.g., thermophiles but not mesophiles), high-temperature germination assays that allow tracking of an exponential increase in signal as spores in marine sediment samples germinate and grow can be used. Distinct exponential increases can be attributed to different populations of germinated thermospores in the post-germination growth phase and are being monitored through measurements of sulfate reduction rates (using radiolabelled 35SO4), strain-specific quantitative PCR (qPCR), and fluorescence in-situ hybridization (FISH) (Rezende et al. 2017). These measurements revealed growth dynamics enabling an estimation of the initial cell numbers using exponential functions. These approaches are being applied to samples obtained via push coring using a remotely operated vehicle at different distances from deep sea hydrocarbon seeps, to test for the presence of abundance gradients of different thermospores. Current results already show a difference in thermospore abundance between sites at different distances from the seep. Quantitative tracking of the dispersal of thermospores by oceanic currents in the deep sea provides an excellent opportunity to investigate their distribution and potential to colonize more remote habitats, improving the understanding of microbial biodiversity and biogeography on our planet. By investigating thermospores and their interactions with the geophysical features of the deep sea and deep subsurface, this study aims to challenge the canonical idea that speciation occurs with distance and presents new perspectives on the theme of microbial biogeography. The evidence supporting the subsurface origin of thermophilic endospores, along with the proposed model for their dispersal and distribution, will contribute toward improving an integrated understanding of ecology and geology and their intersection in the microbial realm.

Preliminary study on the anti-CO2 stress and growth ability of hypsizygus marmoreus mutant strain HY68

BackgroundA high concentration of CO2 will stagnate the development of the newly formed primordia of Hypsizygus marmoreus, hinder the development of the mushroom cap, thereby inhibiting the normal differentiation of the fruiting body. Moreover, in the previous experiment, our research group obtained the mutant strain HY68 of H. marmoreus, which can maintain normal fruiting under the condition of high concentration of CO2. Our study aimed to evaluate the CO2 tolerance ability of the mutant strain HY68, in comparison with the starting strain HY61 and the control strain HY62. We analyzed the mycelial growth of these strains under various conditions, including different temperatures, pH levels, carbon sources, and nitrogen sources, and measured the activity of the cellulose enzyme. Additionally, we identified and predicted β-glucosidase-related genes in HY68 and analyzed their gene and protein structures.ResultsOur results indicate that HY68 showed superior CO2 tolerance compared to the other strains tested, with an optimal growth temperature of 25 °C and pH of 7, and maltose and beef paste as the ideal carbon and nitrogen sources, respectively. Enzyme activity assays revealed a positive correlation between β-glucosidase activity and CO2 tolerance, with Gene14147 identified as the most closely related gene to this activity. Inbred strains of HY68 showed trait segregation for CO2 tolerance.ConclusionsBoth HY68 and its self-bred offspring could tolerate CO2 stress. The fruiting period of the strains resistant to CO2 stress was shorter than that of the strains not tolerant to CO2 stress. The activity of β-GC and the ability to tolerate CO2 were more closely related to the growth efficiency of fruiting bodies. This study lays the foundation for understanding how CO2 regulates the growth of edible fungi, which is conducive to the innovation of edible fungus breeding methods. The application of the new strain HY68 is beneficial to the research of energy-saving production in factory cultivation.

Non-Deterministic Factors Affect Competition Between Thermophilic Autotrophs from Deep-Sea Hydrothermal Vents

Hydrothermal vents provide windows into the rocky subseafloor on Earth and serve as terrestrial analog sites for extraterrestrial environments. By studying patterns of community assembly in hydrothermal vents and using geochemical models, we can better understand how the deep-sea biosphere contributes to local and global biogeochemical cycling and gather valuable information about how similar communities may arise on Earth and beyond Earth. One prevailing thought is that vent microbial community assembly is driven by deterministic factors such as the thermodynamic favorability of redox reactions. We hypothesized that subsurface microbial communities may also be significantly influenced by other factors, such as differential cell yields, varying optimal growth temperatures, and stochasticity. At Axial Seamount in the Pacific Ocean, H2-consuming methanogens of the genera Methanocaldococcus (Topt 82°C) and Methanothermococcus (Topt 65°C) and H2-consuming sulfur reducers of the genus Desulfurobacterium (Topt 72°C) are the most abundant autotrophs that grow optimally at or above 65°C (Fortunato et al. 2017). At one low-temperature hydrothermal vent site, Marker 113, methanogens are the predominant thermophilic autotrophs while at another site, Marker 33, thermophilic autotrophic sulfur reducers predominate. There is no apparent geochemical or thermodynamic explanation for the differences in community composition. In this study, we performed a series of co-culture competition experiments using Methanocaldococcus jannaschii, Methanothermococcus thermolithotrophicus, and Desulfurobacterium thermolithotrophum HR11 as representative methanogens and sulfur reducers common to hydrothermal vents to explain the variations in community composition between thermophilic autotrophs. M. jannaschii increases its cell yield (cells produced per mole of CH4 produced) when grown on very low H2 concentrations as part of a growth rate-growth yield tradeoff (Topçuoğlu et al. 2019). This increase in cell yield could provide methanogens with a competitive growth advantage over H2-consuming sulfur reducers, who otherwise catalyze a more thermodynamically favorable growth reaction. Competition co-culture experiments were conducted between M. jannaschii and D. thermolithotrophum at 72°C and between M. thermolithotrophicus and D. thermolithotrophum at 65°C, both at 1:1 ratios and initial aqueous H2 concentrations of 1.2 mM (high H2) and 85 μM (low H2) to determine the effects of temperature and H2 availability on autotroph competition. For both methanogens, the growth rate, maximum cell concentration, and total CH4 produced decreased when they were grown in co-culture, at low H2, or both relative to monocultures grown with high H2. The methanogen cell yields generally increased in co-culture and at low H2. At both experimental temperatures, the growth rate of D. thermolithotrophum remained unchanged in co-culture and at low H2 relative to monocultures but the maximum cell concentration decreased in co-culture relative to monocultures at both H2 concentrations. However, at low H2, both in mono- and co-culture, there was no detectable H2S produced by the sulfur reducer suggesting a significant shift in growth yield. At both temperatures and H2 concentrations, the sulfur reducer reached higher cell concentrations than the methanogens. Stochasticity or vent fluid chemistry could lead to early colonization of a vent by methanogens followed by niche exclusion of autotrophic sulfur reducers due to a numerical advantage of the methanogens. Therefore, competitive co-culture experiments were run as before at high H2 with varying initial methanogen:sulfur reducer ratios. At 72°C, D. thermolithotrophum reached the same maximum cell concentration and produced the same amount of H2S in monoculture and co-culture even when the methanogens initially outnumbered the sulfur reducer up to 10,000-fold. M. jannaschii reached a lower maximum cell concentration and produced less CH4 in all co-cultures relative to growth in monoculture. At 65°C, D. thermolithotrophum reached the same maximum cell concentrations and produced the same amount of H2S in monoculture and co-culture when the methanogens initially outnumbered the sulfur reducers up to 100-fold. However, when the methanogens initially outnumbered the sulfur reducers 1,000-fold, M. thermolithotrophicus grew as well as in monoculture and the maximum cell concentration and amount of H2S produced by D. thermolithotrophum was significantly lower than in monoculture and the other co-culture conditions. In conclusion, both methanogens and sulfur reducers shift their redox reactions away from CH4 and H2S production, respectively, and towards biomass production when H2 is limiting. This should be accounted for in thermodynamic predictive models. Furthermore, a combination of growth temperatures lower than the optimum of sulfur reducers and high initial methanogen cell concentrations relative to sulfur reducers can lead to a long-term predominance of methanogens over autotrophic sulfur reducers in vent environments through niche exclusion.

Adaptive laboratory evolution of a thermophile toward a reduced growth temperature optimum.

Thermophily is an ancient trait among microorganisms. The molecular principles to sustain high temperatures, however, are often described as adaptations, somewhat implying that they evolved from a non-thermophilic background and that thermophiles, i.e., organisms with growth temperature optima (TOPT) above 45°C, evolved from mesophilic organisms (TOPT 25-45°C). On the contrary, it has also been argued that LUCA, the last universal common ancestor of Bacteria and Archaea, may have been a thermophile, and mesophily is the derived trait. In this study, we took an experimental approach toward the evolution of a mesophile from a thermophile. We selected the acetogenic bacterium T. kivui (TOPT 66°C) since acetogenesis is considered ancient physiology and cultivated it at suboptimal low temperatures. We found that the lowest possible growth temperature (TMIN) under the chosen conditions was 39°C. The bacterium was subsequently subjected to adaptive laboratory evolution (ALE) by serial transfer at 45°C. Interestingly, after 67 transfers (approximately 180 generations), the adapted strain Adpt45_67 did not grow better at 45°C, but a shift in the TOPT to 60°C was observed. Growth at 45°C was accompanied by a change in the morphology as shorter, thicker cells were observed that partially occurred in chains. While the proportion of short-chain fatty acids increased at 50°C vs. 66°C in both strains, Adpt45_67 also showed a significantly increased proportion of plasmalogens. The genome analysis revealed 67 SNPs compared to the type strain, among these mutations in transcriptional regulators and in the cAMP binding protein. Ultimately, the molecular basis of the adaptation of T. kivui to a lower TOPT remains to be elucidated. The observed change in phenotype is the first experimental step toward the evolution of thermophiles growing at colder temperatures and toward a better understanding of the cold adaptation of thermophiles on early Earth.

Characterization, phylogeny and pathogenicity of Fusarium solani causing quinoa basal stem rot in Shanxi Province, China

AbstractA new emerging disease of quinoa, quinoa basal stem rot (QBSR), has been recorded in Shanxi Province, China. Observed during the inflorescence emergence stage, the pathogen causes wilting, foliar chlorosis, leaf abscission and eventual death of quinoa. In this study, isolates were identified as Fusarium solani using morphology, molecular and phylogenetic analysis (using sequences of calmodulin, rDNA internal transcribed spacer region, RNA polymerase II B‐subunits RPB1 RPB1 and RPB2 RPB2, and translation elongation factor 1‐α), and pathogenicity analysis. Twenty‐seven isolates were classified into two different groups according to their morphological characteristics, and the mycelial growth rate of Group 2 was higher than that of Group 1 on six different media. The optimum growth temperature for both groups was 30°C and the highest mycelial growth rates were 9.2 and 10.0 mm/day, for Groups 1 and 2, respectively. The sporodochial conidia (SC) and aerial conidia (AC) germinated normally at 15–25°C and 10–40°C, respectively, and the width of SC and AC germ tubes were 1.4 and 2.2 μm. The lethal temperature for AC was 51°C for 20 min, and SC stopped germinating at 45°C. Pathogenicity tests of representative isolates showed that mycelia or conidia caused typical symptoms of QBSR on wounded basal stems of Chenopodium quinoa at 6 days postinoculation. The optimum temperature for the onset of QBSR was 30°C for both groups of isolates, and lesion lengths ranged from 5.4 to 5.9 cm. Knowledge of this pathogen will provide useful information for effective disease management to prevent its spread.

Validation of competition and dynamic models for Shiga toxin-producing Escherichia coli (STEC) growth in raw ground pork during temperature abuse

Epidemiological evidence suggests that pork products may be a vehicle for STEC transmission to humans. This study was conducted to validate competition and dynamic models for the growth of STEC during simulated temperature abuse of raw ground pork. Maximum specific growth rates μmax were modeled as a function of temperature using the Cardinal parameter equation, and a dynamic model was validated using sinusoidal temperature profiles. The Acceptable Prediction Zone (APZ) method was used to evaluate the model's performance. The competition model was well fitted to the experimental data having 93% (1849/1981) residual errors within the desired APZ. Growth rates were not different between STEC O157 and non-O157; however, serogroup O91 showed two to three times lower μmax than other STEC at 10, 25, and 30 °C. The theoretical minimum and optimum growth temperature for all STEC groups ranged from 3.4 to 7.8 °C and 33–35 °C, respectively. The dynamic model showed good prediction performance (pAPZ = 0.98) with the experimental data. These results can be used to inform risk assessment models and to support the implementation of risk mitigation strategies to improve the microbiological safety of raw pork products.

Identification of a New Compound (4-Fluoro-2-Trifluoromethyl Imidazole) Extracted from a New Halophilic Bacillus aquimaris Strain Persiangulf TA2 Isolated from the Northern Persian Gulf with Broad-Spectrum Antimicrobial Effect.

The unique ecosystem of the Persian Gulf has made it a rich source of natural antimicrobial compounds produced by various microorganisms, especially bacteria, which can be used in the treatment of infectious diseases, especially those of drug-resistant microbes. This study aimed to identify antimicrobial compounds in the bacteria isolated from the northern region of the Persian Gulf in Abadan (Chavibdeh port), Iran, for the first time. Sampling was performed in the fall on November 15, 2019, from 10 different stations (water and sediment samples). The secondary metabolites of all isolates were extracted, and their antimicrobial effects were investigated. 16S ribosomal ribonucleic acid sequencing was used for the identification of the strains that showed the best inhibition against selected pathogens, and growth conditions were optimized for them. A fermentation medium in a volume of 5000 mL was prepared to produce the antimicrobial compound by the superior strain. The extracted antimicrobial compounds were identified using the gas chromatography-mass spectrometry technique. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined for the superior strain. The effects of salinity, pH, and temperature on the production of antimicrobial compounds were determined by measuring the inhibitory region (mm) of methicillin-resistant Staphylococcus aureus (MRSA). Four new strains with antimicrobial properties (i.e., Halomonas sp. strain Persiangulf TA1, Bacillus aquimaris strain Persiangulf TA2, Salinicoccus roseus strain Persiangulf TA4, and Exiguobacterium profundum strain Persiangulf TA9) were identified. The optimum growth temperatures were determined at 37-30, 37, and 40 °C for TA1 and TA2, TA4, and TA9 strains, respectively. The optimum pH values for the four strains were 7, 6-7, 7.5, and 6.5-7.5, respectively. The optimal salt concentrations for the four strains were 15%, 2.5-5%, 7.5%, and 5%, respectively. The ethyl acetate extract of strain Persiangulf TA2 showed extensive antimicrobial activity against human pathogens (75%) and MRSA. The most abundant compound identified in TA2 extract was the new compound 4-fluoro-2-trifluoromethyl imidazole. The MBC and MIC for the ethyl acetate extract of strain TA2 were 20 and 5 mg. mL-1 (Staphylococcus aureus), 40 and 20 mg. mL-1 (MRSA, Escherichia coli, and Enterococcus faecalis), 40 and 10 mg. mL-1 Acinetobacter baumannii), and 80 and 40 mg. mL-1 (Staphylococcus epidermidis, Shigella sp., Bacillus cereus, and Klebsiella pneumoniae), respectively. The optimal conditions for antibiotic production by TA2 strain were 5% salt concentration, pH of 7, and temperature of 35 °C. Newly detected natural compounds in TA2 strain due to superior antimicrobial activity even against MRSA strain can be clinically valuable in pharmacy and treatment.

Rising temperatures can negate CO2 fertilization effects on global staple crop yields: A meta-regression analysis

Many meta-analyses have reported increased seed yields in response to rising carbon dioxide concentrations ([CO2]) or decreased yields in response to warming temperatures, but synthesis of their concurrent effects is lacking. We conducted an extensive meta-regression analysis of wheat, rice, soybean, and maize seed yield responses to simultaneous increases in [CO2] and temperature and examined the sensitivity of potential yields to levels projected for mid- and late-century periods using CMIP6 climate model outputs. Positive linear yield responses to increasing [CO2] were observed in maize, rice, and soy, with slopes ranging from +4 % - (100 µmol mol−1) -1 in rice to +10 % - (100 µmol mol−1) -1 in soy. In contrast, wheat yield response to increasing [CO2] was not linear, instead flattening by +200 µmol mol−1 of CO2 elevation. Negative linear yield responses to warming were modeled for the three cereal crops, with slopes of −7.6% -°C −1 in wheat and −9.5% -°C −1 in rice and maize. Soy yield did not decrease with increasing warming treatments in this analysis, but experimental warming treatments did not exceed optimal soy growth temperature ranges. Across the three crops with available observations, yield responses were significantly lower in Free-Air CO2 Enrichment than in studies using other fumigation methods, likely due to the associated rapid fluctuations in [CO2]. Considering potential future eC and eT levels simultaneously, we find that eC fertilization effects on seed yield will likely be greatly reduced or entirely negated in the major crops maize, rice, and wheat. Net yield responses to concurrent [CO2] and temperature increases should not be based on limited observations of responses to either factor in isolation, because the levels of each factor as well as experimental conditions influence responses.

First Report of Root Rot Caused by Phytophthora lacustris on alder (Alnus lusitanica) in Spain.

Alder decline caused by Phytophthora species is considered one of Europe´s most important diseases of natural ecosystems. In Spain, P. x alni, P. uniformis, P. x multiformis and P. plurivora have been detected in association with root and collar rot in riparian alder populations (Pintos et al. 2010, 2012, 2017; Haque et al. 2014). During the active growth periods 2020-2021, a field survey of the newly designed species Alnus lusitanica (Vít et al. 2017) was carried out at the most symptomatic areas along the Miño-Sil river basin in Galicia and León (NW-Spain). Samples of bark, root, rizosphere soil of declining trees, and river water were collected. Symptoms, similar to those caused by other Phytophthora species, included low leaf density of the canopy, generally with smaller and chlorotic leaves, and branches with dry tips. In some cases, the presence of cankers on the trunk were observed (Figure 1). Necrotic lesions were transferred onto V8-PARPH, a Phytophthora selective medium. Soil and water were baited with carnation petals. Three isolates of one Phytophthora species was recovered: two from the roots of trees, and one from river water at three different sites. The isolates were transferred to V8 agar and incubated a 22ºC in the dark. Colonies had petaloid patterns, and their optimum growth temperature ranged from 25 to 30 °C. In soil extract, non-caducous, non-papillate and ovoid sporangia were produced. Sporangia averaged 41.0×29.4 μm with a length/breadth ratio of 1.39. Internal proliferation occurred (Figure 2). No sexual structures were observed . Genomic DNA was extracted from mycelium obtained from pure cultures of three Phytophthora isolates. The ITS region of the ribosomal DNA template was performed by nested PCR using DC6 (Cooke et al. 2000) and ITS4 (White et al. 1990) in the first round, and ITS6 (Cooke et al. 2000) and ITS4 in the second. The mitochondrial gene cox1 was amplified with primers CoxF4N and CoxR4N (Kroon et al. 2004). BLASTn analyses showed 100% identity to the type of P. lacustris (AF266793, 156 matching bp) for ITS, and 100% identity to the ex-type of P. lacustris (MH136916, 596 matching bp) for the cox1 sequence. Phylogenetic analysis indicated that our isolates were localized in the same evolutionary branch as P. lacustris based on consense sequences of ITS and cox1 sequences. The ITS and cox1 sequences generated in this study were deposited in GenBank with accession number OP588369 and OP548051, and one isolate isolated from root (CECT 21212) was submitted to the Spanish Type Culture Collection (Paterna, Valencia). Pathogenicity tests with isolate CECT 21212 were conducted on ten 3-year-old alders (A. lusitanica) growing in free draining 5 L pots. One shallow cut was made into the cambium at the root collar level. A colonized 5-mm mycelial agar plug, from a 7-day-old culture was inserted into every wound (mycelial surface face-down) and sealed with Parafilm®. Five control plants were inoculated with a sterile agar plug. Plants were kept in a controlled chamber at 25 ºC and 80% humidity. After a 3-week incubation period, inoculated plants showed dieback symptoms and necrosis of the inner bark tissue (Figure 3). Lesion lengths ranged from 2 to 8 cm. Control plants remained symptomless. P. lacustris was recovered from all inoculated plants, but not from the control ones. To our knowledge, this is the first report of P. lacustris causing root rot on alders in Spain. This new detection represents an increased threat to riparian alders by the presence of an additional Phytophthora species associated with alder decline, since P. lacustris can readily adapt to a wide variety of climatic conditions with the ability to infect different hosts (Nechwatal et al. 2013).

Growth kinetic model of Bacillus pasteurii under different pH and temperature conditions in sodium carboxymethyl cellulose solid-free drilling fluid

Increasing the bonding force between broken blocks to form a complete borehole wall is essential to solving borehole wall destabilization in broken strata, and introducing microbial-induced calcite deposition (MICP) technology into the field of drilling fluids establishes a novel research idea for manifesting this technical method. However, during drilling, the pH value and temperature of the drilling fluid constantly change, affecting the activities of microorganisms and the effect of MICP. Therefore, exploring the growth law of microorganisms in drilling fluid environments under different temperatures or pH conditions is necessary. This study used experimental research and theoretical analysis to compare the growth of Bacillus pasteurii in sodium carboxymethyl cellulose (CMC) solid-free and xanthan gum biopolymer solid-free drilling fluid. The growth of B. pasteurii in CMC solid-free drilling fluid in pH = 7.0–9.8 and temperatures from 30 to 50 °C was studied, and the growth kinetic model of B. pasteurii was constructed. The mean square error, decision coefficient (R2), accuracy factor (Af), and deviation factor (Bf) were used to verify the accuracy of the model. CMC solid-free drilling fluid is more suitable for B. pasteurii survival than XG solid-free drilling fluid. The optimum growth temperature of B. pasteurii in CMC solid-free drilling fluid was 32 °C, and the OD600 reached 2.146 at 24 h pH = 8.2 was the optimum pH environment for B. pasteurii in CMC solid-free drilling fluids; the OD600 reached 2.065 at 24 h. As a first-order growth kinetic model, the modified Gompertz model accurately described B. pasteurii growth in CMC solid-free drilling fluid under different temperature strips or pH values. As a secondary growth kinetic model, the square root model accurately described the pattern of pH (7.0–8.2) and temperature effects on the maximum specific growth rate (μmax), with R2 = 0.961, Af = 1.084, Bf = 0.9979, and an error of less than ±0.05. The Arrhenius model accurately described the pattern of pH effects on B. pasteurii (μmax) at pH = 8.6–9.8, with R2 = 0.9996, Af = 1.0036, Bf = 0.999, and an error of less than ±0.02. The effect of temperature was inversely proportional to the growth of B. pasteurii; the higher the temperature, the lesser the B. pasteurii growth. In the pH range of 7.0–8.2, the higher the pH value, the better the B. pasteurii growth; however, at an increased pH range of 8.6–9.8, the higher pH value decreased the B. pasteurii growth. This study is the first to investigate the effect of temperature or pH on B. pasteurii growth in CMC solid-free drilling fluid. Our study provides a scientific basis and theoretical reference for the subsequent research on the induced domestication of B. pasteurii in high-temperature and strong alkaline environments and its engineering application.

Halomonas flagellata sp. nov., a halophilic bacterium isolated from saline soil in Xinjiang.

A Gram-stain-negative, strictly aerobic, motile, slightly curved rod-shaped bacterium with multiple flagella, designated strain EGI 63088T, was isolated from a bulk soil of Kalidium foliatum, collected from Wujiaqu in Xinjiang Uighur Autonomous Region, PR China. The optimal growth temperature, salinity, and pH for strain EGI 63088T growth were 30°C, 3% (w/v) NaCl and 8, respectively. Phylogenetic analysis using 16S rRNA gene sequences indicated that strain EGI 63088T showed the highest sequence similarities to Halomonas heilongjiangensis 9-2T (97.94%), H. lysinitropha 3(2)T (97.51%), and H. daqiaonensis CGMCC 1.9150T (97.08%). The average nucleotide identity and digital DNA-DNA hybridization values between the strain EGI 63088T and H. heilongjiangensis 9-2T were 89.03 and 41.10%, respectively. The DNA G + C content of the genome for strain EGI 63088T was 66.3mol%. The most prevalent antibiotic resistance and virulence-related genes in Halomonas genomes were Streptomyces cinnamoneu EF-Tu mutant, pilT, and cheY, respectively. The predominant fatty acids of strain EGI 63088T were summed feature 8 (C18: 1 ω6c and/or C18: 1 ω7c), summed feature 3 (C16: 1 ω6c and/or C16: 1 ω7c), and C16: 0; its major respiratory quinone was ubiquinone-9 (Q-9), and the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine. According to the above results, strain EGI 63088T is considered a novel species of the genus Halomonas, for which the name Halomonas flagellata sp. nov. is proposed. The type strain is EGI 63088T (= KCTC 92047T = CGMCC 1.19133T).

Molecular Identification and Pathogenicity of Diaporthe eres and D. hongkongensis (Diaporthales, Ascomycota) Associated with Cherry Trunk Diseases in China.

This study aimed to identify fungal species associated with trunk diseases of sweet cherries (Prunus avium) in several commercial cherry orchards in Beijing, Guizhou and Shandong provinces, China. In total, eighteen fungal strains that fitted well into the species concept of Diaporthe were isolated. Based on both morphological and multi-locus phylogenetic analyses of internal transcribed spacer region (ITS), beta-tubulin (tub-2), calmodulin (Cal) and translation elongation factor 1-α (tef1-α) sequencing data, fourteen isolates were identified as Diaporthe eres, while four isolates were classified as D. hongkongensis. Here, we report D. hongkongensis causing sweet cherry branch dieback disease and, further, we confirmed the host association of D. eres with sweet cherries in China. A pathogenicity assay revealed the ability of both D. eres and D. hongkongensis to cause shoot necrosis and stem lesions on Prunus avium cv. 'Brooks' (mean lesion lengths of 1.86 cm and 1.56 cm, respectively). The optimal temperature for the growth of both Diaporthe species was tested. The optimal growth temperature for D. hongkongensis was 30 °C, and the 25-28 °C temperatures were the most favorable for the growth of D. eres strains. This research advances the understanding of fungal trunk diseases in fruit crops, particularly gummosis and branch dieback disease in Chinese cherry orchards, and will aid growers in making decisions about cultural practices and disease management.

Population and Transcriptomic Changes of the Tropical Fish Parasite Caligus confusus (Copepoda: Caligidae) with Seasonal Variations in Sea Temperature

Fish–parasite systems could be subject to two scenarios under climate change: (i) increased water temperature might enhance parasite metabolism, allowing the parasite to spread rapidly; (ii) parasitism could decrease if the optimal temperature for growth and transmission is exceeded. Sea lice are parasitic copepods commonly found on marine fish in tropical regions, yet their biology remains poorly investigated. In this study, we analyzed the changes in infection levels and the transcriptomic response of the tropical sea louse Caligus confusus to two seasonal seawater temperatures (30 °C, “warm”, and 21 °C, “cold”). The prevalence of C. confusus was significantly higher in the colder water. A de novo transcriptomic analysis of C. confusus, the first for a tropical sea louse, revealed 426 over-expressed and 1402 down-expressed transcripts at the lower temperature. In particular, we observed over-expression of transcripts encoding vitellogenins (vit-1, vit-2, vit-4, and vit-6) and matrix metalloproteinases (mmp-2 and mmp-9), which are involved in reproduction and development. These results suggest that the cold tropical season physiologically favors C. confusus and that low temperature favors embryo development, which might ultimately lead to a higher prevalence. It is possible, therefore, that climate change could reduce some tropical sea lice populations during extreme warming events.

Pathogenecity testing of indigenous Bacillus thuringiensis isolates against chickpea pod borer, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) in Ethiopia

Helicoverpa armigera (Hübner) is the most significant insect pest of chickpea in Ethiopia. Sole reliance on insecticide causes both human and environmental health problems. A common soil bacterium, Bacillus thuringiensis used as a bioinsecticide to control numerous Lepidopteran larvae. Therefore, indigenous isolates were collected, screened, tested for pathogenicity against H. armigera with a reference Bacillus. var. thuringiensis. From 121 soil samples collected, 23 Bacillus species were isolated and purified. Bt index ranged from 0.33 to 1.00 and the mean was 0.73 average Bt index. Absorbance values ranged from 0.46 to 0.98. Screening biotoxicity assay was conducted at the rate of 1 mL against 3rd instar larvae of H. armigera. Experiments were replicated three times; ten larvae were used per replicate and reputed thrice. Ten indigenous Bt isolates were screened based on larval mortality which ranged between 27.78% and 75.83% and LT50 values of 45.71–143.9 h. The highest mortality of 75.83% and LT50 of 45.71 h was recorded from reference Bt followed by indigenous Bt isolate KDL with mortality percentage of 69.16% and LT50 of 48.49 h. Statistically no significant difference was observed among the tested bacterial isolates. Biotoxicity assay was conducted at five concentrations ranging from 0.25 to 2 mL. The LD50 and LD 90 values were 0.84–1.24 mL and 1.54–1.98 mL, respectively. The lowest LD50, 90 of 0.84 mL and 1.54 mL were recorded from KDL Bt isolate compared to reference Bt whose LD50 and LD90 were 0.84 mL and 1.66 mL. Results of Bt growth under varied values of temperature ranged from 15oC to 40 °C. All the Bt isolates showed growth at all the values of the tested temperatures with steady growth from 15 °C to 20 °C and uniform growth observed from 20 °C to 30 °C. After 35 °C, the growth of all the Bt isolates declined indicating that 30 °C–35 °C is the optimum growth temperature range. Protein content ranged from 0.43 to 0.97 (mg/mL). However, protein content and larval mortality was not linearly correlated. It was concluded that all the tested indigenous Bacillus isolates were virulent enough and identified as a promising candidate bioinsecticide against H. armigera. Hence, recommended for further intensive evaluations under greenhouse and field trails.

Identification and control of gas-producing bacteria isolated from the swollen bagged soy sauce

The swelling of soy sauce bags seriously affects product quality and causes food safety problems, which has become an urgent problem to solve in the condiment industry. Here, gas-producing bacteria in the swollen bagged soy sauce were isolated and identified to provide an effective control method for inhibiting their growth and solving the swelling of soy sauce bags. It was found that three gas-producing bacteria isolated from the swollen bagged soy sauce were confirmed as Bacillus amyloliquefaciens (G1), Bacillus sp. (G2) and Bacillus subtilis (P3) using 16S rDNA analysis. The strains' morphologies, growth rates, and physiological and biochemical characteristics were also compared. Further studies yielded the optimal growth time, temperature and pH for the three gas-producing bacteria (B. amyloliquefacien: 24 h, 37 °C, and pH 7; Bacillus sp.: 18 h, 30 °C, and pH 6.5–7.5; B. subtilis: 36 h, 30 °C, and pH 8). Bacillus sp. was more salt tolerant than the other two. Then the antibacterial effect of the combination was tested by the physicochemical index. The results showed that filtering through a 0.22 μm inorganic micro-filtration membrane, sterilizing at 121 °C for 2 min, and adding 1 g/kg potassium sorbate was effective methods to inhibit three gas-producing bacteria and control the swelling of soy sauce.

Grapevine dieback caused by Botryosphaeriaceae species, Paraconiothyrium brasiliense, Seimatosporium vitis-viniferae and Truncatella angustata in Piedmont: characterization and pathogenicity

Grapevine Trunk Diseases (GTDs) are major threats in Mediterranean countries, causing economic losses due to reduced grape yields and long-term vine productivity, as well as death of grapevines. A survey was conducted in Piedmont (Northern Italy) during 2021-2022 to investigate the species diversity and distribution of GTD pathogens in this important Italian wine region. Morphological and multi-locus phylogenetic analyses (based on ITS, tef1, tub2, act and rpb2) identified species of Botryosphaeriaceae at high frequency, including Botryosphaeria dothidea, Diplodia mutila, Diplodia seriata and Neofusicoccum parvum. Other pathogens commonly associated with GTDs, including Eutypa lata, Fomitiporia mediterranea and Phaeomoniella chlamydospora, were also isolated. Less commonly isolated species included Neocucurbitaria juglandicola, Paraconiothyrium brasiliense, Seimatosporium vitis-viniferae and Truncatella angustata. Pathogenicity tests with two representative isolates of each species were carried out using one-year-old potted grapevine cuttings (‘Barbera’). All isolates (except N. juglandicola) caused brown wood necrotic vascular discolourations on inoculated plants and were successfully re-isolated. Effects of temperature on colony growth were also assessed. For all tested isolates there was no growth at 5°C, only four isolates (Botryosphaeriaceae) grew at 35°C, and optimum growth temperatures were between 20 and 25°C. This is the first record of Paraconiothyrium brasiliense and Neocucurbitaria juglandicola associated with symptomatic grapevines in Italy.