The optimum and tolerance pH range is correlated to colonial morphology in isolates of the entomopathogenic fungus Beauveria bassiana – a potential biopesticide

Beauveria bassiana is an insect pathogenic fungus used as a biopesticide in crop pest management. It exists saprophytically in the soil. The pH of the soil and the insect cuticle and its haemocoel may influence fungal survival and infection potential. Therefore the tolerance and optimum pH ranges were studied in 29 isolates of B. bassiana. Germination and growth bioassays in liquid culture medium adjusted to pH values in the range 3–14 at unit intervals were studied. A pH of 3 was found to be toxic to all isolates – conidia germinated at this pH but growth was totally inhibited. All isolates tolerated a pH of 5–13. Some isolates showed tolerance to a pH of 4 and/or 14 as well. Sixteen isolates showed a wide range of pH optimum of 5–13 while others had a narrower optimal range. The relation of the pH tolerance and optimum range of an isolate to its phenotypic characters (colonial morphology and growth-rate) was studied. All isolates with a chalky type colonial morphology had an intermediate growth rate and showed a wide optimum range of pH 5–13 or 5–14. All isolates with dusty type colonial morphology were found to have a high growth rate and a wide pH tolerance range of 4–14. Both chalky and dusty colonial morphology are due to a growth pattern characterized by iterated conidiation with a very brief hyphal stage. The correlation observed between these traits based on external phenotype would facilitate prediction of the pH tolerance characters of an isolate of B. bassiana.     

Isolation,identification and characterisation of the nematophagous fungus Arthrobotrys thaumasia (Monacrosporium thaumasium) from China

The objective of this study is to select a native isolate of Arthrobotrys thaumasia (Monacrosporium thaumasium), a nematophagous fungus that shows potential for use in biocontrol programmes for ruminants. First, we looked for native isolates of A. thaumasia and characterised them using light microscopy and molecular markers. Then, we determined the effect of temperature, pH and nutrition on the growth rate and trap formation of a representative isolate. Of the 1532 samples of different types related to sheep and cattle, 11 isolates of A. thaumasia were isolated, and their occurrence frequency in the samples was 0.71% (11/1532). We sequenced the rDNA internal transcribed spacer of isolate NBS005, submitted it to GenBank (ID: KX640093) and then sequenced it using BLAST. The NBS005 could not grow at 37.5°C but could grow from 11°C to 35°C, and it exhibited its optimum growth at 30°C on 1% corn meal agar (CMA). Over 4 days, the fungus did not grow in the pH interval from 1 to 3 or from 13 to 14 but did grow in the pH interval from 4 to 12 and exhibited its optimum growth between pH 9 and 10 on 2% CMA. The factors responsible for the trap formation of NBS005 in liquid culture were identified. Trap formation was induced only by contact with L3 lysate. The concentrations of sucrose, ammonium chloride and tryptone of 0.4, 0.2 and 0.2%, respectively, promoted trap formation, and there were higher numbers of trapping nets in 2% wheat bran liquid medium containing L3 lysate.     

Competition between soil bacteria and fusarium

Summary The phenomenon of competition has been characterized in liquid medium and sterile soil systems using a variety of soil bacteria andFusarium oxysporum f.cubense as test organisms. For most of the bacteria, suppression of the fungus was the result of a biologically induced nitrogen deficiency, this effect being reversed by the addition of excess inorganic nitrogen. High populations of competitors were found in two soils of neutral pH, but no isolates competed in the acid San Alejo loam.Agrobacterium radiobacter was able to compete when San Alejo loam was limed to about pH 6.6. Inhibition of the fungus by a number of gram-positive, spore-forming rods could not be accounted for in terms of competition for nutrients or by antibiotic production in artificial media.The competitive ability ofA. radiobacter when tested in twelve Central American soils was found to be related to pH in acid and neutral environments but was correlated with texture, organic-matter content and total nitrogen in soils of intermediate pH. In all soils where inhibition occurred, the competitive effect was overcome by additions of inorganic nitrogen. Excluding the group ofBacillus spp., the competitive ability of soil bacteria was related to the ability to develop in the absence of amino acids and growth factors but could not be correlated with growth rates of the bacteria in soil or liquid medium.It is suggested that competition for nutrients is a significant means of ecological control among members of the soil microflora and, together with competitive interactions for space and oxygen, may be the major factors governing the biological control of soil-borne fungi.The investigation was supported in part by a grant from the United Fruit Company. Agronomy Paper No. 471.     

Impact of nutritional supplements and monosaccharides on growth, oxalate accumulation, and culture pH by Sclerotinia sclerotiorum

Sclerotinia sclerotiorum D-E7 was studied to determine the impact of nutritional supplements and monosaccharides on growth, oxalate accumulation, and culture pH in broth media (initial pH c. 5). Cultures with 0.1% nutritional supplement (tryptone, yeast extract, or soytone) yielded minimal growth, 2-3 mM oxalate, and a final culture pH of 4.2-4.8. In contrast, cultures with 0.1% nutritional supplement and 25 mM glucose yielded significant growth, minimal oxalate (<1 mM), and a final culture pH of 2.8-3.7. Similar trends were observed when glucose in 0.1% soytone cultures was replaced with 25 mM d-mannose, l-arabinose, or d-xylose. With 1% soytone-25 mM glucose cultures, growth and oxalate accumulation ( approximately 21 mM) occurred with little change in initial pH. This was not the case with 1% soytone-250 mM glucose cultures; increased glucose levels resulted in a decrease in oxalate accumulation ( approximately 7 mM) and in final culture pH (3.4). Time-course studies with these cultures revealed that oxalate accumulation was suppressed during growth when the culture pH dropped to <4. Overall, these results indicate that (1) the decrease in external pH (i.e. acidification) was independent of oxalate accumulation and (2) acidification coupled to glucose-dependent growth regulated oxalate accumulation by Sclerotinia sclerotiorum.     

Distribution of <Emphasis Type="Italic">Rigidoporus lignosus</Emphasis> genotypes in a rubber plantation,as revealed by somatic compatibility

A study was conducted to determine the spatial distribution of somatic incompatibility groups (SIGs) in Rigidoporus lignosus populations responsible for white root disease in a south Sumatera rubber plantation. Most isolates colonizing the stumps were somatically compatible with isolates from either a nearby stump or a living rubber tree, which suggested evidence of stump-to-tree or stump-to-stump clonal growth of the fungus. SIGs of R. lignosus occupied an area territorially in which the minimum size estimate for the largest genotype was 25m in length.     

The effect of copper on the growth of wood-rotting fungi and a blue-stain fungus

The effect of copper (II) ions on the growth of three brown-rot fungi, six white-rot fungi and one blue-stain fungus in solid medium was evaluated. The fungi were grown in malt extract agar with different concentrations of copper added, and the radial growth rate was determined. At the end of the incubation period, the mycelial biomass and the media pH were determined. The white-rot and blue-stain fungus grew up to 3 mM and 6 mM copper, respectively and the brown-rot fungi were the only ones that grew up to 10 mM, with higher growth rates than those shown by the other fungi. In general, the brown-rot fungi produced greater acidification in the culture media than the white-rot fungi and blue-stain fungus, and the acidification increased when the amount of copper was increased. The biomass production for the different species, in the absence or presence of copper, was not related to the radial growth rate, and the fungal species that produced the greatest biomass amounts did not correspond to those that presented the highest growth rates. The brown-rot fungi Wolfiporia cocos and Laetiporus sulfureus and blue-stain fungus Ophiostoma sp. demonstrated greater tolerance to high copper concentrations in solid medium than the white-rot fungi, determined as radial growth rate. On the other hand, the highest biomass producers in solid medium with copper added were the white-rot fungi Ganoderma australe and Trametes versicolor and the brown-rot fungus Gloeophyllum trabeum.     

Acidification effects on vesicular-arbuscular mycorrhizal (VAM) infection, growth and nutrient uptake of established heathland herb species

A greenhouse experiment was carried out to determine whether the decline of Arnica montana L. in heathland vegetation in the Netherlands could be caused by a detrimental effect of soil acidification on vesicular-arbuscular mycorrhiza of this species. Arnica montana and two non-declining species from the same habitat, Hieracium pilosella L. and Deschampsia flexuosa (L.) Trin., were grown with and without the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus fasciculatum (Thaxter sensu Gerdemann) Gerd. and Trappe in pots with an extremely nutrient-poor, sandy soil. They were percolated weekly with nutrient solution with different pH values, viz. 5.5, 4.5, 3.5 and 2.5. At intervals of three weeks and up to 12 weeks, measurements were made on growth, nutrient uptake and VAM infection.In the most acid treatments growth and nutrient uptake were reduced in all species. VAM infection decreased only slightly with decreasing pH of the treatments. Without VAM, Arnica montana died and Hieracium pilosella hardly grew at the most acid treatments. Therefore it is concluded that VAM decreased the stress caused by the most acid treatments. Leachate from the most acid treatment had a pH of approximately 4, and contained considerable amounts of aluminium, dissolved from the solid phase of the soil. This might have played a role in the detrimental effects on the plants in the case of the most acid treatment. No evidence was found in this experiment that the decline of Arnica montana was due to detrimental effects of soil acidification on VAM of this species.     

Growth and emergence of the stonefly Leuctra nigra in coniferous forest streams with contrasting pH

1. Growth and emergence of the acidification tolerant stonefly Leuctra nigra were studied in four coniferous forest streams with contrasting pH. Invertebrate samples were taken bimonthly during May 1997 to April 1998. Emerging L. nigra were captured in two of the streams during April to July 1998 using pyramidal emergence traps. In addition, a laboratory experiment was conducted to evaluate growth of L. nigra in response to variations in food quality (neutral or acid conditioned leaves) and water pH. 2. The invertebrate community in low pH streams had lower diversity and density compared with neutral streams. Abundance of L. nigra was also lower, despite the dominance of this stonefly in the invertebrate community in the acidic streams. 3. Secondary production of L. nigra was 2.04 and 3.07 g DW m^–2 year^–1 in the neutral streams compared with 1.02 and 1.27 g DW m^–2 year^–1 in the acidic streams. The P/B ratio differed only slightly among streams. 4. More adult L. nigra emerged in the neutral stream compared with the acidic stream, despite the density of the pre‐emerging fifth instar nymphs of L. nigra being similar in the two streams. Adult size (expressed as wing length) and female fitness were also significantly greater in the neutral stream. 5. Laboratory growth of L. nigra nymphs was significantly lower when fed acid conditioned alder leaves rather than neutral conditioned leaves. There was no difference in growth when nymphs were fed neutral conditioned leaves in acid and neutral water. 6. Our study shows multiple, sub‐lethal effects on life history of the acidification tolerant stonefly L. nigra in coniferous streams with low pH. The results suggest that these effects could be mediated primarily by low food quality. These findings add to the existing evidence that coniferous plantation forestry has a negative impact on stream ecosystems.     

Physiological characterisation of Colletotrichum gloeosporioides,the incitant of anthracnose disease of noni in India

Ten isolates of Colletotrichum gloeosporioides were collected from different noni growing areas of Tamil Nadu, Karnataka and Kerala in India and their pathogenicity was proved under glass house conditions. Effect of different pH levels, temperature, light intensity and media were tested against the growth of C. gloeosporioides under in vitro. The results indicated that the growth of C. gloeosporioides was maximum in pH range of 6.50–7.00 and temperature range of 25–30°C. Exposure of the fungus to alternate cycles of 12 h light and 12 h darkness resulted in the maximum mycelial growth of C. gloeosporioides compared to the 24 h exposure to continuous light and 24 h exposure to continuous dark. Among the different media tested, host leaf extract medium supported significantly the maximum growth of all the 10 isolates of C. gloeosporioides followed by potato dextrose agar. Further, the strains were found to vary morphologically between the isolates under the study.     

Characterization of maize endosperm-derived suspension cells throughout the culture cycle and enhancement of tissue friability

Batch suspension cultures derived from developing maize (Zea mays L.) endosperm were examined throughout the culture cycle to determine the interaction between the tissue and the medium in relation to sugar transport and the effect of subculturing procedures on growth and friability. The growth rate and friability were improved by increasing the frequency of subculture or by physical screening during transfer. An increase in the conductivity of the medium preceded a decrease in fresh weight associated with tissue senescence. Sucrose in the medium was rapidly hydrolysed, and fructose was depleted more rapidly than glucose. Tissue sucrose concentration expressed on a dry weight basis was higher during the middle of the growth cycle, but hexose, starch, zein, lipid, and soluble protein levels changed very little. The medium pH declined from 5.2 to about 4.5 within one day of subculture. Medium pH changed to 4.5 within one day regardless of initial pH (3.0 to 7.0), indicating regulation of external pH rather than passive acidification. Results are consistent with studies of sugar uptake by these cultures, and indicate that cell clump size can be manipulated without exogenous auxin. The characterization of this tissue line establishes its suitability as a model system for studies of sugar transport and other biochemical events in developing maize endosperm.     

PHOTOSYNTHESIS OF ALGAL CULTURES AND PHYTOPLANKTON FOLLOWING AN ACID pH SHOCK1

In acidifying lakes, pH decreases abruptly in response to acid precipitation events. We tested the hypothesis that, in comparison to a circumneutral lake, phytoplankton photosynthesis in an acidifying lake is less sensitive to a rapid decrease in pH (acid pH shock). Phytoplankton in Plastic Lake, which is undergoing acidification, was characterized by a predominance of Pyrrophyta, and phytoplankton photosynthesis decreased to a lesser extent in response to an acid pH shock than the photosynthesis of populations from St. Nora Lake, a circumneutral lake located nearby, in which Pyrrophyta were not abundant. Rates of phytoplankton photosynthesis in acid pH shock experiments were significantly correlated with hydrogen ion but not with dissolved inorganic carbon (DIC) concentrations. Depression of photosynthesis following an acid pH shock occurred in axenic cultures of Chlorella pyrenoidosa Chick but was not observed in axenic cultures of the acidophilic alga Chlorella saccharophila (Krug.) Nadson or in three species isolated from Plastic Lake. However, the three isolates were not acidophilic during growth. We conclude that phytoplankton in acidifying lakes consists predominantly of species which are tolerant to acid pH for short periods (hours) but cannot grow at these pHs.     

Carbon regulation of environmental pH by secreted small molecules that modulate pathogenicity in phytopathogenic fungi

Fruit pathogens can contribute to the acidification or alkalinization of the host environment. This capability has been used to divide fungal pathogens into acidifying and/or alkalinizing classes. Here, we show that diverse classes of fungal pathogens—Colletotrichum gloeosporioides, Penicillium expansum, Aspergillus nidulans and Fusarium oxysporum—secrete small pH‐affecting molecules. These molecules modify the environmental pH, which dictates acidic or alkaline colonizing strategies, and induce the expression of PACC‐dependent genes. We show that, in many organisms, acidification is induced under carbon excess, i.e. 175 mm sucrose (the most abundant sugar in fruits). In contrast, alkalinization occurs under conditions of carbon deprivation, i.e. less than 15 mm sucrose. The carbon source is metabolized by glucose oxidase (gox2) to gluconic acid, contributing to medium acidification, whereas catalysed deamination of non‐preferred carbon sources, such as the amino acid glutamate, by glutamate dehydrogenase 2 (gdh2), results in the secretion of ammonia. Functional analyses of Δgdh2 mutants showed reduced alkalinization and pathogenicity during growth under carbon deprivation, but not in high‐carbon medium or on fruit rich in sugar, whereas analysis of Δgox2 mutants showed reduced acidification and pathogencity under conditions of excess carbon. The induction pattern of gdh2 was negatively correlated with the expression of the zinc finger global carbon catabolite repressor creA. The present results indicate that differential pH modulation by fruit fungal pathogens is a host‐dependent mechanism, affected by host sugar content, that modulates environmental pH to enhance fruit colonization.     

Heavy Metal Resistance of the Extreme Acidotolerant Filamentous Fungus Bispora sp

To obtain information on the importance of membrane and zeta potentials as repelling or facilitating forces during the uptake of cationic trace elements, the heavy metal content and the growth resistance of the acidotolerant fungus Bispora. sp. to heavy metals were compared at pH 1.0 and pH 7.0. Cu, Co, Ni, Cd, Cr, and La contents of the fungus were significantly lower at pH 1.0 than at pH 7.0. A similar pH effect occurred with cationic macro elements such as Na, Mg, Ca, Fe, and Mn. Only K and Zn exhibited higher levels at pH 1.0 in the fungus than at pH 7.0. Macro and micro elements present in the medium in anionic form (sulfate, chloride) showed the opposite pattern to cations: Contents were higher at pH 1.0 than at pH 7.0. Minerals present at pH 1.0 predominantly in the electrical neutral, protonated form (phosphate, borate) exhibited a similar cell content at both acid and neutral pH (P) or a higher content at neutral pH than at acid pH (B). The resistance of fungal growth to the cations Cu, Zn, Ni, Co, Cr, and Cd was significantly higher at pH 1.0 than at pH 7.0. Such a difference was not observed with Hg, present in the medium at both pH values as electrically silent HgCl₂. The anionic tungstate exhibited the opposite pattern to cationic heavy metals: The resistance of growth was higher at pH 7.0 than at pH 1.0. A greater growth resistance to heavy metals was correlated with a lower uptake of these elements, and vice versa; Uptake of heavy metals correlated with a lower resistance of fungal growth to these elements. The results are in agreement with the hypothesis that membrane and zeta potentials of the fungus are important factors controlling the uptake of heavy metals and thereby the resistance of growth to these elements: At pH 1.0 positive potentials of fungal hyphae impede the uptake of cationic heavy metals, but facilitate the uptake of anionic species. At neutral pH values the negative potentials facilitate the uptake of cations, but impede the uptake of anions.     

Growth-promoting factors for yeast cells ofParacoccidioides brasiliensis

Low-density seedings of yeast cells ofParacoccidioides brasiliensis give poor growth (as assessed by plating efficiency test) on conventional mycological agar media, and therefore growth-promoting factors for this fungus were sought. Water-extracts of yeast cells of sixP. brasiliensis isolates were all considerably effective in promoting the growth of low-density seedings ofP. brasiliensis isolates Pb-18 and Hachisuga, but had little effect on isolate Bt-4. Horse serum, at a concentration range of 2–4%, moderately or considerably promoted the growth of theseP. brasiliensis isolates. Combinations of the fungus cell extracts with horse serum were highly effective in promoting the growth of all of the fungal isolates. The fungus cell extracts showed siderophore (microbial iron carrier) activity. An iron-chelator, ethylenediaminetetraacetic acid, at a concentration of 100 μM also highly promoted the growth of the fungal isolates in the presence of horse serum, and ferric ion added to culture medium was considerably effective in the growth promotion. These results suggest that deficient utilization of external iron by the fungus cell is one of the growth-limiting processes for low-density seedings of yeast cells ofP. brasiliensis on conventional mycological agar media.     

Phosphate uptake and polyphosphate metabolism of mycorrhizal and nonmycorrhizal roots of pine and of Suillus bovinus at varying external pH measured by in vivo 31P-NMR

 Comparative in vivo ³¹P-NMR analyses of mycorrhizal and nonmycorrhizal roots of Pinus sylvestris and the fungus of Suillus bovinus in pure culture were used to investigate alterations in phosphate metabolism due to changes in external pH in the range 3.5–8.5. All control samples maintained a constant pH in both cytoplasm and vacuole. Mycorrhizal roots and pure fungus, but not nonmycorrhizal roots, transformed accumulated inorganic phosphate into mobile polyphosphate with a medium chain length. Phosphate uptake rates and polyphosphate accumulation responded differently to external pH. In all cases, maximal phosphate uptake occurred at an external pH close to 5.5. At an external pH of 8.5, both roots and fungus showed a distinct lag in phosphate uptake, which was abolished when the external pH was lowered to 7.5. An irreversible effect on phosphate uptake as a consequence of variation in external pH was also observed. The central role of the fungus in regulating mycorrhizal phosphate metabolism is discussed. Accepted: 15 April 1997     

Anaerobic Growth of Candida albicans Does Not Support Biofilm Formation Under Similar Conditions Used for Aerobic Biofilm

C. albicans is an opportunistic fungus causing life-threatening systemic infections particularly in immunocompromised individuals. The organism is a commensal in humans and grows either aerobically, e.g., the oral cavity, or anaerobically, e.g., the gut. We studied anaerobic growth of C. albicans in a defined yeast nitrogen base dextrose medium after adaptation and subculturing in an anaerobic chamber. At 37°C in suspension culture, much slower growth was observed anaerobically with a generation time of 248 min compared to 98 min for aerobic growth. Although the organism grew well on solid medium, shaking increased the growth rate in suspension culture at 37°C. Growth was enhanced at acidic pH compared to neutral or alkaline pH. Cells grown anaerobically produced hyphae, but did not produce biofilm on plastic surface or denture acrylic under either static conditions or with mild shaking, conditions that support aerobic biofilm formation.     

Morphological,cultural and pathogenic characteristics of Coniothyrium zuluense isolates from different plantation regions in South Africa

Coniothyrium canker caused by Coniothyrium zuluense, is a serious stem canker disease of Eucalyptus species in sub-tropical regions of South Africa. This disease is typified by necrotic bark lesions that coalesce to form large kino-impregnated cankers along the stems of trees. The strategy currently used to manage Coniothyrium canker in plantations is to deploy Eucalyptus species or clones that are resistant to the disease. Considerable success has already been achieved in this regard, but the long-term durability of resistance is of concern. Thus, forest managers are interested in the genetic diversity of the pathogen and its potential to overcome disease resistance in planting stock. In this study, 344 isolates of C. zuluense from different plantation regions in South Africa were compared on the basis of colony colour, conidial morphology, growth characteristics on agar and pathogenicity to a susceptible E. grandis clone. Conidia of all C. zuluense isolates measured were similar in size and shape. The fungus is slow growing in culture, which is indicative of its apparent biotrophic habit, with optimum growth observed at 30 °C. Isolates of C. zuluense displayed considerable variation in colony colour and pathogenicity in inoculation trials. Variation in morphology and pathogenicity amongst isolates suggests that C. zuluense has been present in South Africa for an extended period of time, or that it is changing rapidly due to strong directional selection pressures. This revised version was published online in June 2006 with corrections to the Cover Date.     

Soluble phosphate accumulation by Aspergillus niger from fluorapatite

Summary In order to determine conditions that may provide greater solubilization of insouluble phosphate, the fungus Aspergillus niger was grown in a stationary culture containing modified citrate medium supplemented with 800 mg fluorapatite per litre. Solubilization of insouluble phosphate increased with fungal growth, reaching a maximum after 11 days of culture. Soluble phosphate levels were correlated with pH of the culture medium but not with titratable acidity values, probably due to the metabolic activity of the fungus resulting from consumption of sugar in the culture medium. Fructose, glucose, xylose, and sucrose were the carbohydrates that favoured fluorapatite solubilization the most when compared with galactose and maltose. Although increasing fructose concentrations in the culture medium favoured mycelial growth, increased total acidity and a fall in pH, soluble phosphate levels were reduced, probably owing to consumption by the rapidly growing fungus. Among the nitrogen sources tested, ammonium salts favoured the production of larger amounts of soluble phosphate than organic nitrogen (peptone or urea) or nitrate, corresponding to the lowest pH and highest titratable acidity values obtained.     

Community‐level effects of rapid experimental warming and consumer loss outweigh effects of rapid ocean acidification

Climate change and consumer loss simultaneously affect marine ecosystems, but we have limited understanding of the relative importance of these factors and the interactions between them. Moreover, effects of environmental change are mediated by organism traits or life histories, which determine their sensitivity. Yet, trait‐based analyses have rarely been used to understand the effects of climate change, especially in the marine environment. Here we used a five‐week mesocosm experiment to assess the single and interactive effects of 1) rapid ocean warming, 2) rapid ocean acidification, and 3) simulated consumer loss, on the diversity and composition of macrofauna communities in eelgrass Zostera marina beds. Experimental warming (ambient versus + 3.2°C) and loss of a key consumer (the omnivorous crustacean, Gammarus locusta) both increased macrofauna richness and abundance, and altered overall species trait distributions and life history composition. Warming and consumer‐loss favored poorly defended epifaunal crustaceans (tube‐building amphipods), and species that brood their offspring. We suggest these organisms were favored because warming and consumer‐loss caused increased metabolism, food supply and, potentially, settling substrate, and lowered predation pressure from the omnivorous G. locusta. Importantly, we found no single, or interactive, effects of the rapid ocean acidification (ambient versus ?0.35 pH units). We suggest this result reflects natural variability in the native habitat and, potentially, the short duration of the experiment: organisms in these communities routinely experience rapid diurnal pH fluctuations that exceed the mean ocean acidification predicted for the coming century (and used in our experiments). In summary, our study indicates that macrofauna in shallow vegetated ecosystems will be significantly more affected by rapid warming and consumer diversity loss than by rapid ocean acidification.