Comparison of Soil Receptivity to Thielaviopsis basicola, Aphanomyces euteiches, and Fusarium solani f. sp. pisi Causing Root Rot in Pea

ABSTRACT Soil receptivity as a quantifiable characteristic ranging from conduciveness to suppressiveness to soilborne pea pathogens Thielaviopsis basicola and Aphanomyces euteiches was determined by analysis of differences in disease response curves obtained by artificial introduction of inoculum into natural field soil samples. Several parameters, including maximum root rot severity, the area under the health index curve, scores on the first axis of a principal component analysis (PCA) on dose responses, and Weibull model fitting were used to describe the disease responses. In all cases, the Weibull model gave satisfactory fits. PCA yielded a first axis that comprised 86% of the variance found when using Weibull predicted responses for T. basicola and 74% of the variance found for A. euteiches. This PCA axis essentially represented the average increase in disease severity due to the addition of increasing doses of inoculum to the soil. The Weibull scale parameter B, which represents the amount of inoculum necessary to increase root rot severity by 63% with respect to the level caused by pathogens naturally present in the soil, is another means of quantifying the receptivity of soils to these plant pathogens. Weibull parameter B, maximum root rot severity, the areaunder the health index curve, and the scores on the first PCA axis were strongly correlated for each of the pathogens tested individually. To compare the extent and behavior of soil receptivity responses to different pathogens, Weibull parameters B and C (slope at dose B) were chosen because of their universal definition, in contrast to PCA scores. Comparison of the average levels of Weibull parameters B and C indicated significant differences between the pathogens. Yet, no significant similarity in the ranking of the soils was found for the three pathogens, demonstrating that individual soils may interact with different pathogens in totally different ways. In general, soils were suppressive to T. basicola but conducive to A. euteiches, whereas their response to Fusarium solani f. sp. pisi ranged from conducive to suppressive. Therefore, risk assessment of soils prior to planting may require different strategies for each pathogen. Bioassays with soil samples taken before the last pea crop in 1987 and 1991 revealed a significant increase in the natural inoculum potential of soils that mainly was accounted for by A. euteiches and Pythium spp. These results strongly indicate that A. euteiches must be considered one of the most threatening pathogens to pea crops in the Netherlands.     

Interactions between the biocontrol agent Pseudomonas fluorescens CHA0 and Thielaviopsis basicola in tobacco roots observed by immunofluorescence microscopy

Pseudomonas fluorescens CHA0 protects plants from damage caused by several soilborne fungi. In this work, immunofluorescence microscopy was used to investigate the colonization of tobacco roots by CHA0 and its physical relationship with the black root rot fungus Thielaviopsis basicola . The pseudomonad colonized the rhizoplane shortly after planting of tobacco seedlings in sterile soil microcosms, in which it had been introduced as soil inoculant. CHA0 was found between and inside cells in the epidermis and the cortex, as well as in the xylem vessels, within 4–7 days after planting of seedlings. The presence of CHA0 delayed the colonization of the interior of tobacco roots by T. basicola compared with the treatment in which only the fungus had been inoculated. Likewise, the pseudomonad reduced the extent of black root rot from 82% to 28%. However, CHA0 was seldom found in contact with the mycelium of T. basicola or in its vicinity, indicating that direct colonization of the mycelium of T. basicola by CHA0 was not required for protection of tobacco against black root rot. Overall, the results suggest that the interior of the root is a key site for implementation of the strain's biocontrol activity against soilborne plant-pathogenic fungi.     

Effect of pea plant architecture on spatiotemporal epidemic development of ascochyta blight (Mycosphaerella pinodes) in the field

The effect of pea canopy structure on epidemics of Mycosphaerella pinodes was investigated in four spring pea cultivars (Bridge, Obelisque, Solara and Athos) in two field experiments. These cultivars had similar levels of susceptibility to ascochyta blight and presented different architectural features (branching, standing ability and stem height). Stem height ranged from 77 to 95 cm in 1999 and from 72 to 92 cm in 2000, while leaf area index (LAI) ranged from 3·8 to 5·1 in 1999 and from 3·8 to 4·7 in 2000. Internode lengths varied from 4·6 to 6·0 in 1999 and from 3·8 to 4·7 in 2000. Mean distance between nodes in the canopy (MDN) ranged from 12·2 to 15·3 cm in 1999 and from 11·2 to 13·9 cm in 2000. Canopies with different architecture differed in disease progression on stipules and mainly on pods. The levels of disease on stipules and pods were strongly correlated. Moreover, if disease was considered as a function of stipule height up the stem, large differences in vertical disease distribution were observed between cultivars. Three architectural features acted on disease development: cumulative LAI above the node on which disease was assessed (LAIcum) and large MDN favouring disease development, and large internode length reducing disease severity. Modifying LAI distribution and plant organization could be one way to reduce the impact of ascochyta blight, by direct or indirect effects on environmental and dispersal conditions.     

Dynamics of soilborne Rhizoctonia solani in the presence of Trichoderma harzianum: effects on stem canker, black scurf and progeny tubers of potato

Stem canker and black scurf are diseases of potato caused by the fungus Rhizoctonia solani . Spatiotemporal experimentation and empirical modelling were applied for the first time to investigate the effect of antagonistic Trichoderma harzianum on the dynamics of soilborne R. solani on individual potato plants. Trichoderma harzianum reduced the severity of symptoms, expressed as 'rhizoctonia stem lesion index' (RSI), during the first 7 days post-inoculation when the inoculum of R. solani was placed at certain distances (30–60 mm) from the host. For example, with inoculum at 40 mm from the host, RSI was 6 and 40 with and without T. harzianum , respectively. At later observation times, the antagonistic effect was overcome. Trichoderma harzianum reduced the severity of black scurf on progeny tubers. Furthermore, the mean number of progeny tubers per potato plant was reduced by the biocontrol treatment (means of 6·5 ± 1·1 and 9·9 ± 2·7 tubers per plant with and without T. harzianum , respectively), as was the proportion of small (0·1–20·0 g) tubers (48% and 66% with and without T. harzianum , respectively). Additionally, there were fewer malformed and green-coloured tubers in pots treated with T. harzianum than in those without T. harzianum .     

Effect of Heterodera cajani on biomass and grain yield of pigeon pea on vertisol in pot and field experiments

Glasshouse and field trials were conducted to determine the effects of the cyst nematode, Heterodera cajani on biomass and grain yield of pigeon pea, Cajanus cajan. Shoot length, fresh and dry shoot masses, leaf area and pod yields of pigeon pea were significantly reduced by H. cajani. In glasshouse pot experiments, an initial density of 1.0 juveniles per cm³ soil caused 14 to 24% reduction in plant height, root and shoot mass and leaf area. Application of carbofuran 3G (1·5, 3·0 and 6·0 kg a.i./ha) in H. autoclaved soil in pots did not improve growth of pigeon pea; however, its application (6 kg a.i./ha) in H. cajani -infested fields reduced the nematode density ( P < 0·05) and improved plant growth and yield. The densities of eggs and juveniles of H. cajani were 72 and 48% lower in the carbofuran-treated plots than in the control plots 35 and 52 days, respectively, after sowing. No such differences were observed at 70 days after sowing. The tolerance limit for pod yield in field experiments was 2·6 eggs and juveniles of H. cajani per cm³ soil at sowing time. Grain yield was 20 to 25% higher in the carbofuran-treated plots than in the control plots. Application of carbofuran protected the roots from nematode damage during the early stages of plant growth and resulted in good plant growth and yield.     

Effect of Temperature on and Histopathology of the Interaction Between Meloidogyne incognita and Thielaviopsis basicola on Cotton

ABSTRACT Controlled environments were used to study the relationship between the root-knot nematode (Meloidogyne incognita) and Thielaviopsis basicola on cotton. Temperature treatments were continuous 20, 24, and 28 degrees C or two cyclic linear regimes with ranges of 14 to 32 or 18 to 28 degrees C over 24 h. Cotton seeds were planted in fumigated soil infested with T. basicola, M. incognita, or both. After 42 days, pathogen effects on plant growth and pathogen development were evaluated. Histology was conducted on roots collected 14, 28, and 42 days after planting in the continuous 24 degrees C treatment. Reductions in plant height-to-node ratio and total fresh weight were observed for soils infested with both pathogens compared with the control or with soils infested with either pathogen, except for M. incognita-infested soil at 28 degrees C. T. basicola reduced root galling and reproduction of the nematode at all temperatures. Vascular discoloration caused by T. basicola was greater in the presence of M. incognita compared with that by T. basicola alone. At 2 and 4 weeks, histological studies showed that plants grown in all T. basicola-infested soils contained chlamydospore chains on the root surface and in cortical cells. The fungus was not observed inside the vascular cylinder. Roots from 4-week-old plants from soils infested with T. basicola and M. incognita showed fungal sporulation in vascular tissue and localized necrosis of vascular tissue adjacent to the nematodes. At 6 weeks, plants grown in soil infested with T. basicola alone exhibited no remaining cortical tissue and no evidence of vascular colonization by the fungus. Six-week-old plants grown in T. basicola + M. incognita-infested soils exhibited extensive vascular necrosis and sporulation within vascular tissue. These studies suggest that coinfection expands the temperature ranges at which the pathogens are able to cause plant damage. Further, M. incognita greatly increases the access of T. basicola to vascular tissue.     

Relation between cropping frequency of peas and other legumes and foot and root rot in peas

The relation between the frequency of legume crops in a rotation and the root rot severity in pea was examined in a field survey. Additionally, greenhouse experiments were performed with soil samples from legume rotation trials or from farmers' fields. The frequency of pea crops in current rotations proved to be much less than the recommended value of one in six years. The correlation between pea root rot and the number of years that pea or other legumes were not grown on the field under consideration (called crop interval) was weak. Root rot severity correlated better with the frequency of peas or legumes in general over a period of 18 years, but the frequency still explained only a minor fraction of the variation in disease index. Some experimental data pointed to the occurrence of a highly specific pathogen microflora with continuous cropping of only one legume species, but this phenomenon probably does not occur in farmers' fields. In field samples, root disease index for pea correlated well with that for field bean. The survival of resting structures of pathogens such asAphanomyces euteiches probably explains why the frequency of legume cropping has a higher impact than crop interval on root disease incidence. Pea-free periods and legume frequencies have a poor predictive value for crop management purposes.     

烟草根黑腐病菌致病力分化及品种抗性差异研究

 烟草根黑腐病由根串珠霉（Thielaviopsis basicola (Berk.et Br.)Ferr.）引起,主要为害烟株的根部,可使根部组织呈特异性黑色坏死而导致烟苗死亡或地上部分生长不良。该病是世界性的烟草病害,在各产烟国家如美国、日本、加拿大等国普遍发生,在我国主要分布于河南、山东、安徽、云南等地     

The recent history of Puccinia striiformis f.sp. tritici in Denmark as revealed by disease incidence and AFLP markers

Disease observations and amplified fragment length polymorphism (AFLP) markers were used to study recent developments in the Puccinia striiformis f.sp. tritici population in Denmark. The fungus appeared spontaneously at 10 locations in Denmark in 1997 after it was not observed under natural conditions in 1996. The pattern of disease development and prevailing winds suggested that the fungus reappeared by airborne spores from the south or west. In 1998, disease incidence was more evenly distributed throughout the country. Forty-eight single lesion isolates were collected from most crops where the disease was observed in these years; all except one from 1997 belonged to two pathotypes that were not previously detected in the country, and both possessed the newly discovered Yr17 virulence. The isolates were characterized with AFLP markers together with 28 isolates representing eight of 13 pathotypes observed prior to 1996. Initial screening of 240 Pst I/ Mse I AFLP primer combinations on four isolates showed that a primer combination, on average, revealed 0·4 polymorphisms between any isolate pair. A selection of 21 primer combinations resulted in 28 AFLP markers, which revealed 16 AFLP phenotypes among all 76 isolates. The two Yr17- virulent pathotypes consisted of three AFLP phenotypes, which were observed in both 1997 and 1998; the two most frequent AFLP phenotypes occurred at most sampling locations and often within the same crop. AFLP diversity was larger among samples collected prior to 1996, and also in this period most AFLP phenotypes were observed at different sampling locations. These results are consistent with the features of an entirely asexually reproducing pathogen dispersed by aerial spores across large areas.     

Assessing mango anthracnose using a new three-dimensional image-analysis technique to quantify lesions on fruit

An accurate image-analysis method was developed to assess quantitatively the spot-like lesions on fruits resulting from pathogen attack. The technique was applied to evaluation of the development and severity of anthracnose of mango fruit, caused by the fungus Colletotrichum gloeosporioides . In this method, a stepper motor rotates the mango fruit along its longitudinal axis while acquiring a sequence of 360 images of its total surface (one image for each degree). This set of images is used to create a pseudocylindrical 'equal-area' projection of the fruit in a two-dimensional map containing complete morphometrical and photometrical information of its surface. The lesion area can easily be evaluated from this map with image-analysis procedures. Quantitative data (percentage of area affected) can be used to establish an assessment scale for the disease based on lesion spots measured, as well as for detailed laboratory studies of mango anthracnose development. The average error of the method is −0·1%, standard deviation 0·44 ( r ² = 0·99), and it may be adapted for use with most commercial image analysers and for other diseases with spot-like symptoms.     

土壤中烟草根黑腐病菌的实时定量PCR检测技术研究

 Thielaviopsis basicola is a soil-borne plant pathogen which causes root rot disease in tobacco plants. Detection and monitoring of T. basicolain soil is of great significance to control this disease. Based on the differences in internal transcribed spacer (ITS) sequences of T. basicola and other fungal pathogens, a specific primer pair Tb1/Tb2 for T. basicolawas developed. The results showed that the primer pair gave a single amplicon of 330 bp from T. basicola and revealed no undesirable cross-reaction with other seven soil-borne pathogen isolates and three tobacco rhizosphere dominant fungi isolates. With a series of 10-fold genomic DNA dilutions of T. basicola, the detection limit of 1 pg/μL in conventional PCRand100 fg/μL in real-time quantitative PCR was achieved. With DNA from the soil inoculated with different numbers of T. basicola conidia, the detection limit was 10 conidia per reaction in conventional PCR and 0.4 conidia per reaction in real-time quantitative PCR.     

Response of interspecific Brassica juncea/Brassica rapa hybrids and their advanced progenies to Albugo candida (white blister)

Transfer of factors for resistance to white blister disease caused by Albugo candida between Brassica species involving two genotypes each of B. juncea and B. rapa was studied in hybrids. More hybrids were obtained by in vivo than in vitro techniques, although an in vitro phase was a prerequisite for the establishment of in vivo hybrids. Hybrids were identified by PCR-based inter-simple sequence repeat (ISSR) markers with both male and female species-specific bands being identified. There was a positive correlation between disease severity and number of days after sowing ( r  > 0·93), the highest being towards pod formation and plant maturity at 110 days after sowing. The plants from F₂ and BC₁ progeny showed higher resistance to A. candida than either of the parents. Plants of B. juncea and B. rapa with high field resistance (disease index < 1·0) were selected from BC₂ and F₂BC₁ generations. The frequency of plants classified as resistant in BC₂ progeny ranged from 4·5 to 39·0% in cross-combinations involving B. juncea genotypes as female parent, compared with 100% in the reciprocal cross involving B. rapa as female parent.     

Leaf scald (Xanthomonas albilineans) incidence and its effect on yield in seven sugarcane cultivars in Guadeloupe

Seven 5—month-old sugarcane cultivars difTering in resistance to leaf scald disease were inoculated by the decapitation technique with Xanthomonas albilineans. The effects of disease progress and incidence on yields were studied for the plant (first harvest) and two ratoon crops (second and third harvest). The percentage of diseased stalks and disease severity at first harvest 5 months after inoculation were 0·7 and 0·4, respectively, for the most resistant cultivar and 71·0% and 63·3, respectively, for the most susceptible cultivar. They decreased in all cultivars in both ratoon crops, but were still important in one cultivar (B69379). Significant ( P = 0·05) yield reductions of 12% and 21% occurred in two of the seven cultivars (B69566 and B69379, respectively). The number of symptomatic sugarcane stalks in the first ratoon crop (second harvest) was lower than the number of stalks colonized by the pathogen. Symptoms and yield losses of cultivars R570 and B69566 varied with the crop. Yield losses occurred in cultivar R570 only in the plant crop when this cultivar displayed numerous symptoms. Cultivar B69566 appeared to recover from the disease to a certain extent from the plant to the second ratoon crop (third harvest), as did the resistant cultivars in the first ratoon crop. In contrast, severe leaf scald symptoms were observed in the case of cultivar B69379 regardless of the crop, and significant yield losses occurred in the two ratoon crops. These results support the recommendation that cultivar B69379 should not be replanted in Guadeloupe.     

Comparison of three diagrammatic keys for the quantification of late blight in tomato leaves

Three diagrammatic grading keys were designed for the assessment of the severity of late blight (caused by Phytophthora infestans ) in tomato leaves. Simplified and broad keys considered, respectively, six (3, 12, 22, 40, 60 and 77%) and eight (3, 6, 12, 22, 40, 60, 77 and 90%) levels of disease severity, whilst a modified key based on a previous proposal for potato late blight considered six levels (1, 5, 10, 16, 32 and 50%). The keys were validated by 24 evaluators who assessed digital images of tomato leaves exhibiting different areas with lesions. Evaluator errors were compared using a mixed model in which evaluators were considered as random effects and the keys and evaluations as fixed effects. The accuracy and precision of the evaluators were compared by simple linear regression between the estimated and actual values of disease severity. The repeatability of evaluators was assessed using Pearson's correlation coefficient. There was significant ( P  ≤   0·001) variability amongst the errors made by evaluators, although the precision of each of the three keys was high with a coefficient of determination (R²) of 0·96, 0·93 and 0·83 for the simplified, broad and modified key, respectively. Repeatability of estimations amongst the evaluators was adequate (correlation coefficients of 0·91, 0·91 and 0·90 for the three keys, respectively). The simplified and broad keys resulted in higher precision and accuracy for the estimation of severity than did the modified key. Since the simplified key considers a smaller number of disease severity levels, its use is recommended in the assessment of late blight in tomato leaves.     

Differential Sensitivity of Phytophthora parasitica var. nicotianae and Thielaviopsis basicola to Monomeric Aluminum Species

ABSTRACT Aluminum (Al) is toxic to many plant pathogens, including Thielaviopsis basicola and Phytophthora parasitica var. nicotianae. Because fungi-toxicity of Al has been described in soils over a wide pH range, multiple species of Al may be responsible for pathogen suppression. The goals of this work were to determine the sensitivity of T. basicola and P. para-sitica var. nicotianae to Al over a range of pH values, quantify the toxicity of monomeric Al species to production of sporangia of P. parasitica var. nicotianae and chlamydospores of T. basicola, and detect the accumulation of Al in pathogen structures. A complete factorial treatment design was used with Al levels ranging from 0 to 100 muM and pH levels ranging from 4 to 6 in a minimal salts medium. The chemistry of test solutions was modeled using GEOCHEM-PC. Colonies were grown in 5% carrot broth, and after 1 or 2 days, the nutrient solution was removed, colonies were rinsed with water, and Al test solutions were added to each of four replicate plates. After 2 days, propagules were counted and colonies were stained with the Al-specific, fluorescent stain lumogallion. The oomycete P. parasitica var. nicotianae was sensitive to multiple monomeric Al species, whereas sensitivity of T. basicola to Al was pH-dependent, suggesting that only Al(3+) is responsible for suppression of this fungal pathogen. Chlamydospore production by T. basicola was inhibited at pH values <5.0 and Al levels >20 muM, whereas sporangia production by P. parasitica was inhibited at Al levels as low as 2 muM across all pH values tested. The lumogallion stain was an effective technique for detection of Al in fungal tissues. Aluminum accumulated in sporangia and zoospores of P. parasitica var. nicotianae and in nonmelanized chlamy-dospores of T. basicola, but not in cell walls of either organism. The differential sensitivity of the two organisms may indicate that true fungi respond differently to Al than members of the oomycota, which are more closely related to plants.     

Bioassay to assess root rot in pea and effect of root rot on yield

Infection of pea roots by soil-borne pathogens causes foot and root rot. In 1985 research was started to develop a method to predict the root rot likely to occur in prospective pea fields. In a bioassay the pea cultivar Finale was sown in a composite soil sample from each field in pots under standardized conditions in the greenhouse. The plants were removed at the green bud stage and the severity of root rot recorded. Between 1985 and 1988 approximately 200 field pea crops were monitored for root rot development. Forty-eight fields were bioassayed in 1986, 51 in 1987 and 30 in 1988. Each year, root rot readings in the bioassay and disease severity readings at field sampled plants at flowering and green pod were linearly correlated (P<0.001). As the degree of root rot in the field crop increased, there was a proportional lower yield. In heavily infested fields, up to a 50% yield reduction occurred.The bioassay in pots proved to be a reliable method for predicting root rot severity in sampled pea fields.     

Susceptibility of invasive taxa of European blackberry to rust disease caused by the uredinial stage of Phragmidium violaceum under field conditions in Australia

European blackberry ( Rubus fruticosus agg.) is an aggregate of closely related taxa, with at least 15 taxa naturalized in Australia. Biological control of this Weed of National Significance, using the nonindigenous rust fungus Phragmidium violaceum , is effective when the weather is conducive to multiple cycles of infection, but some blackberry taxa escape severe disease. Thirty-one taxa of naturalized R. fruticosus agg. from southeastern Australia were isolated, their DNA phenotype determined and clones of each taxon inoculated with P. violaceum isolate SA1. Disease development was monitored for at least four generations of uredinia on large potted plants under field conditions. Although variation in mean disease severity appeared continuous over the range of Rubus clones tested, counts of uredinia and telia enabled identification of eight resistant taxa. Fine scale variation in susceptibility to rust disease was observed when different clones of R. leucostachys with the same DNA phenotype were found to express either resistance or susceptibility to P. violaceum (SA1). There were significant differences among 23 Rubus taxa rated as susceptible to rust disease in the mean number of leaves emerging per latent period of uredinia (LELPU). Mean LELPU appeared to account for some of the variation in two measures of mean disease severity observed among susceptible Rubus clones, although the correlation was insignificant (0·10 <  P  > 0·05).     

Interaction of root-lesion nematodes, Pratylenchus thornei and P. crenatus, with the fungus Thielaviopsis basicola and a grey sterile fungus on roots of pea (Pisum sativum)

Pratylenchus thornei invaded excised pea roots in agar in greater numbers and penetrated the cortex more deeply than P. crenatus . Both species fed on the roots ectoparasitically and displaced root cells into the surrounding medium. The cytoplasm of cortical cells near cither nematode became granulated, with enlarged vacuoles and nuclei. P. thornei also caused these responses in the endodermis. Infection of the root surface with a grey sterile fungus inhibited invasion by P. crenatus and P. thornei . Infection by Thielaviopsis basicola inhibited P. thornei invasion but encouraged penetration by P. crenatus and the hyphae were found deeper in the cortex when P. crenatus was present.     

Effects of root- and stolon-infecting fungi on root-colonizing nematodes of white clover

The effects on white clover ( Trifolium repens ) of different combinations of the nematodes Meloidogyne trifoliophila , Helicotylenchus dihystera and Heterodera trifolii and nine stolon-infecting and three root-infecting fungi were studied in a glasshouse experiment. The presence of the fungus Phytophthora megasperma alone increased ( P  < 0·001) root-rot severity and reduced ( P  < 0·001) plant growth. Other species combinations, such as Phoma nebulosa and Alternaria alternata , interacted and increased root-rot severity. Combinations of P. megasperma with Pythium irregulare , and P. nebulosa with Phoma medicaginis or A. alternata , increased M. trifoliophila populations. Several other fungi ( P. irregulare , P. nebulosa , Colletotrichum coccodes , Macrophomina phaseolina , P. medicaginis and Phoma sp.) interacted with the nematode M. trifoliophila causing severe root-knot symptoms. The results indicated that fungi and nematodes interacted to cause root and stolon rot and reduced yields, and that poor persistence of white clover in pastures is likely to be a problem with a complex etiology.     

Effects of nitrogen, phosphorus, potassium and calcium nutrition on strawberry anthracnose

The effects of a range of concentrations of four nutrients – nitrogen, phosphorus, potassium and calcium – in fertilizer solutions on the severity of anthracnose on strawberry cv. Nyoho cultivated under a noncirculation hydroponics system were determined after inoculation with Colletotrichum gloeosporioides . Crop growth and tissue nitrogen, phosphorus, potassium and calcium contents of the entire above-ground parts of the plant were also investigated. Elevated nitrogen and potassium concentrations in the fertilizer solution increased disease severity in contrast to phosphorus and calcium. Treatment with either NH₄ or NO₃ nitrogen was not significantly different. The dry weight of the strawberry plants increased significantly with elevated concentrations of nitrogen ( R ² = 0·9078) and phosphorus ( R ² = 0·8842), but was not influenced by the elevated amounts of potassium ( R ² = 0·8587) and calcium ( R ² = 0·6526) concentrations.