Response of Nitrosospira sp. strain AF-like ammonia oxidizers to changes in temperature, soil moisture content, and fertilizer concentration

Very little is known regarding the ecology of Nitrosospira sp. strain AF-like bacteria, a unique group of ammonia oxidizers within the Betaproteobacteria. We studied the response of Nitrosospira sp. strain AF-like ammonia oxidizers to changing environmental conditions by applying molecular methods and physiological measurements to Californian grassland soil manipulated in the laboratory. This soil is naturally high in Nitrosospira sp. strain AF-like bacteria relative to the much-better-studied Nitrosospira multiformis-like ammonia-oxidizing bacteria. Increases in temperature, soil moisture, and fertilizer interacted to reduce the relative abundance of Nitrosospira sp. strain AF-like bacteria, although they remained numerically dominant. The overall abundance of ammonia-oxidizing bacteria increased with increasing soil moisture and decreased with increasing temperature. Potential nitrification activity was altered by interactions among temperature, soil moisture, and fertilizer, with activity tending to be higher when soil moisture and temperature were increased. The increase in potential nitrification activity with increased temperature was surprising, given that the overall abundance of ammonia-oxidizing bacteria decreased significantly under these conditions. This observation suggests that (i) Nitrosospira sp. strain AF-like bacteria may respond to increased temperature with an increase in activity, despite a decrease in abundance, or (ii) that potential nitrification activity in these soils may be due to organisms other than bacteria (e.g., archaeal ammonia oxidizers), at least under conditions of increased temperature.     

Phylogeny of nitrite reductase (nirK) and nitric oxide reductase (norB) genes from Nitrosospira species isolated from soil

Ammonia-oxidizing bacteria are believed to be an important source of the climatically important trace gas nitrous oxide (N(2)O). The genes for nitrite reductase (nirK) and nitric oxide reductase (norB), putatively responsible for nitrous oxide production, have been identified in several ammonia-oxidizing bacteria, but not in Nitrosospira strains that may dominate ammonia-oxidizing communities in soil. In this study, sequences from nirK and norB genes were detected in several cultured Nitrosospira species and the diversity and phylogeny of these genes were compared with those in other ammoniaoxidizing bacteria and in classical denitrifiers. The nirK and norB gene sequences obtained from Nitrosospira spp. were diverse and appeared to be less conserved than 16S rRNA genes and functional ammonia monooxygenase (amoA) genes. The nirK and norB genes from some Nitrosospira spp. were not phylogenetically distinct from those of denitrifiers, and phylogenetic analysis suggests that the nirK and norB genes in ammonia-oxidizing bacteria have been subject to lateral transfer.     

Adaptation of ammonia-oxidizing microorganisms to environment shift of paddy field soil

Adaptation of microorganisms to the environment is a central theme in microbial ecology. The objective of this study was to investigate the response of ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) to a soil medium shift. We employed two rice field soils collected from Beijing and Hangzhou, China. These soils contained distinct AOB communities dominated by Nitrosomonas in Beijing rice soil and Nitrosospira in Hangzhou rice soil. Three mixtures were generated by mixing equal quantities of Beijing soil and Hangzhou soil (BH), Beijing soil with sterilized Hangzhou soil (BSH), and Hangzhou soil with sterilized Beijing soil (HSB). Pure and mixed soils were permanently flooded, and the surface-layer soil where ammonia oxidation occurred was collected to determine the response of AOB and AOA to the soil medium shift. AOB populations increased during the incubation, and the rates were initially faster in Beijing soil than in Hangzhou soil. Nitrosospira (cluster 3a) and Nitrosomonas (communis cluster) increased with time in correspondence with ammonia oxidation in the Hangzhou and Beijing soils, respectively. The 'BH' mixture exhibited a shift from Nitrosomonas at day 0 to Nitrosospira at days 21 and 60 when ammonia oxidation became most active. In 'HSB' and 'BSH' mixtures, Nitrosospira showed greater stimulation than Nitrosomonas, both with and without N amendment. These results suggest that Nitrosospira spp. were better adapted to soil environment shifts than Nitrosomonas. Analysis of the AOA community revealed that the composition of AOA community was not responsive to the soil environment shifts or to nitrogen amendment.     

Molecular analysis of ammonia-oxidising bacteria in soil of successional grasslands of the Drentsche A (The Netherlands)

Changes in the community structure of chemolitho-autotrophic ammonia-oxidising bacteria of the beta-subgroup Proteobacteria were monitored during nutrient-impoverishment management of slightly acidic, peaty grassland soils, which decreased in pH with succession. Specific PCR, cloning and sequence analysis, denaturing gradient gel electrophoresis (DGGE) and probe hybridisation were used to analyse rDNA sequences directly recovered from successional soils. Four previously characterised ammonia oxidiser sequence clusters were recovered from each soil, three associated with the genus Nitrosospira and one with the genus Nitrosomonas. All samples were dominated by Nitrosospira-like sequences. Nitrosospira cluster 3 was the most commonly recovered ammonia oxidiser group in all fields, but a greater representation of Nitrosospira clusters 2 and 4 was observed in older fields. Most probable number (MPN) counts were conducted using neutral and slightly acid conditions. Neutral pH (7.5) MPNs suggested a decrease in ammonia oxidiser numbers in later successional fields, but this trend was not observed using slightly acid (pH 5.8) conditions. Analysis of terminal MPN dilutions revealed a distribution of sequence clusters similar to direct soil DNA extractions. However, an increased relative recovery of Nitrosospira cluster 2 was observed for acid pH MPNs compared to neutral pH MPNs from the most acidic soil tested, in agreement with current hypotheses on the relative acid tolerance of this group.     

祁连山北坡青海云杉林下苔藓层对土壤水分空间差异的影响

苔藓层是青海云杉林(Picea crassifolia forest)下的一个重要层片,它通过截持降水和减少土壤蒸发,对土壤水分产生影响。以祁连山北坡排露沟小流域的一个阴坡(海拔2700 m,植被为青海云杉林)作为样坡,于2010—2012年生长季对青海云杉林下有、无苔藓层覆盖地点的土壤水分进行对比观测。结果表明在祁连山青海云杉林下,苔藓层覆盖能减少土壤水分的空间差异。主要表现为:(1)无苔藓覆盖各观测点土壤含水量极差达62.2 mm,空间变异系数(CV)为17.3%;有苔藓层覆盖观测点土壤含水量空间变异系数为2.3%,仅为无苔藓覆盖地点的1/7.5,空间差异极显著(sig.0.001)。(2)在持续无雨、小雨后和连阴雨天气下,有苔藓覆盖地点土壤含水量空间差异均显著小于无苔藓覆盖地点(sig.0.05)。在持续无雨的情况下,无苔藓层覆盖各观测点土壤含水量空间变异系数平均值为19.8%,有苔藓覆盖地点土壤含水量空间变异系数平均值为6.6%,仅为无苔藓覆盖地点的1/3。小雨后,无苔藓覆盖各观测点土壤含水量空间变异系数平均值为15.2%,有苔藓覆盖地点土壤水分空间变异系数平均为5.1%,为无苔藓覆盖地点的1/3。连阴雨后,无苔藓覆盖各观测点土壤含水量空间变异系数平均为15.4%,有苔藓覆盖地点土壤水分空间变异系数平均为4.6%,为无苔藓覆盖地点的1/3.3。(3)持续无雨的情况下,苔藓层减小土壤水分空间差异的作用主要反映在土壤表层0—15 cm,对深层的作用不显著。而小雨和连阴雨后,苔藓层对15—80 cm深层土壤影响显著,而表层0—15 cm没有明显规律。     

Species Diversity of Uncultured and Cultured Populations of Soil and Marine Ammonia Oxidizing Bacteria

Although molecular techniques are considered to provide a more comprehensive view of species diversity of natural microbial populations, few studies have compared diversity assessed by molecular and cultivation-based approaches using the same samples. To achieve this, the diversity of natural populations of ammonia oxidising bacteria in arable soil and marine sediments was determined by analysis of 16S rDNA sequences from enrichment cultures, prepared using standard methods for this group, and from 16S rDNA cloned from DNA extracted directly from the same environmental samples. Soil and marine samples yielded 31 and 18 enrichment cultures, respectively, which were compared with 50 and 40 environmental clones. There was no evidence for selection for particular ammonia oxidizer clusters by different procedures employed for enrichment from soil samples, although no culture was obtained in medium at acid pH. In soil enrichment cultures, Nitrosospira cluster 3 sequences were most abundant, whereas clones were distributed more evenly between Nitrosospira clusters 2, 3, and 4. In marine samples, the majority of enrichment cultures contained Nitrosomonas, whereas Nitrosospira sequences were most abundant among environmental clones. Soil enrichments contained a higher proportion of identical sequences than clones, suggesting laboratory selection for particular strains, but the converse was found in marine samples. In addition, 16% of soil enrichment culture sequences were identical to those in environmental clones, but only 1 of 40 marine enrichments was found among clones, indicating poorer culturability of marine strains represented in the clone library, under the conditions employed. The study demonstrates significant differences in species composition assessed by molecular and culture-based approaches but indicates also that, employing only a limited range of cultivation conditions, 7% of the observed sequence diversity in clones of ammonia oxidizers from these environments could be obtained in laboratory enrichment culture. Further studies and experimental approaches are required to determine which approach provides better representation of the natural community.     

The Nitrifying Bacteria in Soils from Rothamsted Classical Fields and Elsewhere

S ummary . Ammonia oxidizing autotrophic nitrifying bacteria in soil samples from 3 classical Rothamsted fields and elsewhere were counted by a modified 2-layered silica gel plating method. Counts ranged from 0-18000/g of dry soil, the maximum number being found in the Broadbalk farmyard manure plot. Nitrosomonas europaea was isolated in pure culture and detected only in soils treated with dung or other organic fertilizer. Nitrosocystis coccoides and Nitrosospira spp. were found in other soils. Nitrobacter spp. were present in many soils and of the 18 pure cultures isolated there seemed to be 3, or possibly 4, different colony types.     

Predicting live herbaceous moisture content from a seasonal drought index

In order to provide a relatively simple means of predicting live herbaceous plant moisture content from a readily available meteorological index with an accuracy adequate for fire hazard assessment, the moisture content fluctuations of certain species were correlated with the values of a seasonal drought index based on soil moisture deficiency. The simple linear regression models provided the best fit of the relationship between plant moisture content and the Keetch-Byram drought index (KBDI) values. KBDI predicted with accuracy, for two growing seasons, the moisture content of three annual herbaceous plants ( Piptatherum miliaceum, Parietaria diffusa, Avena sterillis) with shallow rooting systems, typical of the understory vegetation of Pinus brutia forests in the Mediterranean region of Crete, Greece. The greatest aberrations between measured and predicted values of plant moisture content were observed early (May) and late (September) in the growing season, when plant phenology (flushing and withering stages respectively) appears to become the dominant factor in determining plant moisture content regardless of the soil moisture conditions. The KBDI was poorly correlated with the live-needle moisture content of deeply rooted P. brutia trees and modestly with the soil water content of the upper layers. This indicates that the index adequately reflects the moisture condition of the surface soil layers but not the water content deeper in the soil.     

三北防护林地区土壤水分动态研究

东北西部干旱、半干旱地区影响农业、畜牧业发展的关键因子之一是水分。水分在这一地区变化规律是５～６月春旱阶段,７．８、９三个月是土壤水分丰盛期,各层次水分相差不大,１０月至来年２月是土壤储水期。农田防护林的建立减少了蒸发,提高了土壤水分。但距林带不同距离调节水分幅度不同,明显出现水分最佳区和水分低谷区。根据这一规律可以调整种植结构,充分合理的利用土壤水分,为推广节水型农业提供了途径。防护林带的种类、树高、树种及林带间的距离对林网内水分的分配都有较大影响,这一结论为防护林体系的优化提供了科学依据。     

甘肃敦煌西湖荒漠-湿地生态系统土壤水分空间异质性及其影响因子研究

土壤水分是内陆荒漠区湿地生态系统中重要的限制因子,为了揭示该区域土壤水分空间分布特征,采用传统统计学和地统计学相结合的方法,对甘肃敦煌西湖国家级自然保护区0~200cm内各层土壤水分的空间变异性及海拔、土壤质地和植被对其的影响进行了研究,旨在为极干旱区湿地生态系统植被修复和保育提供科学依据。结果表明:(1)本研究所得各变量的变异系数、块金方差、基台值、变程和结构比分别为36.51%~88.65%、0.007~0.098、0.112~0.549、116~453和76.6%~97.6%,各变量均为中等变异,存在高度异质性,具有较强空间自相关。(2)深层(60~200cm)土壤水分含量较浅层(0~60cm)变异大,且不同层次土壤水分含量的空间异质性差别也较大,空间变异主要发生在较小尺度上(分维数D在1.902~1.989之间)。(3)海拔是影响该区域深层土壤水分空间变异的主导因子。(4)土壤质地与浅层(0~60cm)土壤水分含量的相关性大于与深层(60~200cm)土壤含水量的相关性,它们与海拔相关性表现相反;草本植被盖度与浅层土壤水分含量呈较高的正关联关系,灌木根量与深层土壤水分含量呈较高的负关联关系。     

Response of Nitrosospira sp. Strain AF-Like Ammonia Oxidizers to Changes in Temperature, Soil Moisture Content, and Fertilizer Concentration

Very little is known regarding the ecology of Nitrosospira sp. strain AF-like bacteria, a unique group of ammonia oxidizers within the Betaproteobacteria. We studied the response of Nitrosospira sp. strain AF-like ammonia oxidizers to changing environmental conditions by applying molecular methods and physiological measurements to Californian grassland soil manipulated in the laboratory. This soil is naturally high in Nitrosospira sp. strain AF-like bacteria relative to the much-better-studied Nitrosospira multiformis-like ammonia-oxidizing bacteria. Increases in temperature, soil moisture, and fertilizer interacted to reduce the relative abundance of Nitrosospira sp. strain AF-like bacteria, although they remained numerically dominant. The overall abundance of ammonia-oxidizing bacteria increased with increasing soil moisture and decreased with increasing temperature. Potential nitrification activity was altered by interactions among temperature, soil moisture, and fertilizer, with activity tending to be higher when soil moisture and temperature were increased. The increase in potential nitrification activity with increased temperature was surprising, given that the overall abundance of ammonia-oxidizing bacteria decreased significantly under these conditions. This observation suggests that (i) Nitrosospira sp. strain AF-like bacteria may respond to increased temperature with an increase in activity, despite a decrease in abundance, or (ii) that potential nitrification activity in these soils may be due to organisms other than bacteria (e.g., archaeal ammonia oxidizers), at least under conditions of increased temperature.     

三种干旱指数在干旱区沼泽湿地土壤水分遥感反演中的应用

湿地水文条件对湿地生态系统结构和功能起着关键作用。借助卫星遥感技术,以干旱区疏勒河中下游沼泽湿地为研究对象,基于Landsat8 OLI遥感影像的温度植被干旱指数(TVDI)、垂直干旱指数(PDI)和归一化干旱监测指数(NPDI)3种干旱监测方法,结合野外湿地实测土壤水分数据进行比较和验证。结果表明:(1)TVDI、PDI和NPDI3种干旱指数在不同土层中均与土壤实测水分呈显著负相关(P0.01),且NPDI指数与表层土壤水分的拟合效果最好。不同土层中,3种干旱指数与土壤水分的拟合优度大小均为NPDITVDIPDI;(2)研究区同一湿地类型不同土层的土壤水分存在显著差异(P0.05),且不同湿地类型的土壤水分总体上表现为随着土壤深度的增加呈递增态势;(3)研究区同一土层不同湿地类型的土壤水分差异显著(P0.05),且不同土层中土壤水分的大小均为:沼泽化草甸草本沼泽季节性咸水沼泽内陆盐沼。     

太湖流域丘陵区两种土地利用类型土壤水分分布控制因素

为探究太湖流域丘陵区典型土地利用类型(如竹林地和茶园)土壤水分的控制因素,在不同深度土壤水分定期观测的基础上,根据前7d降雨量将研究时段划分为干旱状态和湿润状态,利用分类与回归树(CART)方法得出不同干湿状态下土壤水分分布的主控因子,并借助典范对应分析(CCA)定量分析不同土地利用类型、不同土壤深度土壤水分格局与环境因子关系。结果表明:(1)高程、土地利用类型和土层厚度对土壤水分分布的相对贡献率最大,但在不同干湿状态下其影响程度存在差异;(2)干旱状态时土壤水分主要受高程、坡度、地形湿度指数(TWI)和剖面曲率等地形因素的作用,而土层厚度和粘粒也分别为0—20 cm和20—40 cm深度土壤水分的主控因子;(3)在湿润状态下,茶园0—20 cm土壤水分的主控因素为地形因子,在20—40 cm则以土壤性质为主,竹林地两个深度的土壤水分受地形和土壤性质的作用都很强,其中20—40 cm深度土壤水分与环境因子的关系较0—20 cm深度更为复杂。     

Patterns of community change among ammonia oxidizers in meadow soils upon long-term incubation at different temperatures

The effect of temperature on the community structure of ammonia-oxidizing bacteria was investigated in three different meadow soils. Two of the soils (OMS and GMS) were acidic (pH 5.0 to 5.8) and from sites in Germany with low annual mean temperature (about 10 degrees C), while KMS soil was slightly alkaline (pH 7.9) and from a site in Israel with a high annual mean temperature (about 22 degrees C). The soils were fertilized and incubated for up to 20 weeks in a moist state and as a buffered (pH 7) slurry amended with urea at different incubation temperatures (4 to 37 degrees C). OMS soil was also incubated with less fertilizer than the other soils. The community structure of ammonia oxidizers was analyzed before and after incubation by denaturing gradient gel electrophoresis (DGGE) of the amoA gene, which codes for the alpha subunit of ammonia monooxygenase. All amoA gene sequences found belonged to the genus Nitrosospira. The analysis showed community change due to temperature both in moist soil and in the soil slurry. Two patterns of community change were observed. One pattern was a change between the different Nitrosospira clusters, which was observed in moist soil and slurry incubations of GMS and OMS. Nitrosospira AmoA cluster 1 was mainly detected below 30 degrees C, while Nitrosospira cluster 4 was predominant at 25 degrees C. Nitrosospira clusters 3a, 3b, and 9 dominated at 30 degrees C. The second pattern, observed in KMS, showed a community shift predominantly within a single Nitrosospira cluster. The sequences of the individual DGGE bands that exhibited different trends with temperature belonged almost exclusively to Nitrosospira cluster 3a. We conclude that ammonia oxidizer populations are influenced by temperature. In addition, we confirmed previous observations that N fertilizer also influences the community structure of ammonia oxidizers. Thus, Nitrosospira cluster 1 was absent in OMS soil treated with less fertilizer, while Nitrosospira cluster 9 was only found in the sample given less fertilizer.     

黄土丘陵区小流域土壤水分时空变异

采用定位监测法,对2002—2005年黄土丘陵区小流域土壤水分的时空动态规律及其主要影响因素进行了分析.结果表明,研究区年均土壤水分剖面呈倒“L”型,水分最低点出现在1.0 m深处.表层（0～0.2 m）和下层（2.4 m以下）土壤水分年际间的差异达显著水平,而季节间的显著差异则表现在土壤表层和中层（1.0～2.4 m）;干旱年的降水不足显著提高了中、下层土壤水分的空间变异性,而丰水年的降水补充作用则显著降低了上层土壤水分的空间变异性,对中、下层土壤水分变异的影响不大.坡向对土壤水分变异的影响最大,其次是土地利用类型,坡位的影响最小.在干旱年或每年雨季开始时,坡向对土壤水分的影响随土层的加深而减弱,土地利用类型的影响则随之增强;在丰水年或每年雨季结束时,土地利用类型只对上层土壤水分状况具有显著影响,且其影响程度小于坡向的影响.     

西安附近苹果林地的土壤干层

根据西安附近苹果林下土壤含水量测定,研究了0~6m之间土壤含水量的变化与土壤干层问题。资料表明,西安附近15龄苹果林下2~3.5m深处土壤含水量为9.1%~9.2%,形成了发育弱的长期性土壤干层,10龄苹果林地2~4m深处也有干层发育,表明黄土高原的土壤干层分布已达黄土高原南部的关中地区;6龄苹果林下土壤有干化的显示,但无干层发育。分析得出,由降水少量决定的、埋藏深度小而厚度大的薄膜水带的存在是引起土壤干层发育的直接作用的因素。土壤干层的出现会引起土壤与植被的退化,应当避免严重的土壤干层出现。     

沙地土壤种植杨树-刺槐混交林后持水特性变化的研究

通过对比法研究了沙地土壤种杨树刺槐混交林后持水特性的变化。结果表明,沙地土壤种植杨树,刺槐纯林和混交林后,土壤持水特性,孔隙分布状况和水容量得到了改善和提高,提高和改善的幅度均以混交林最大,无林地植被覆盖后土壤持水性能改善的根本原因在于土壤有机质含量的增加,从而改善了土壤的孔隙状况。     

Changes in the community structure of ammonia-oxidizing bacteria during secondary succession of calcareous grasslands

The community structure of β-subclass Proteobacteria ammonia-oxidizing bacteria was determined in semi-natural chalk grassland soils at different stages of secondary succession. Both culture-mediated (most probable number; MPN) and direct nucleic acid-based approaches targeting genes encoding 16S rRNA and the AmoA subunit of ammonia monooxygenase were used. Similar shifts were detected in the composition of the ammonia oxidizer communities by both culture-dependent and independent approaches. A predominance of Nitrosospira sequence cluster 3 in early successional fields was replaced by Nitrosospira sequence cluster 4 in late successional fields. The rate of this shift differed between the two areas examined. This shift occurred in a background of relative stability in the dominant bacterial populations in the soil, as determined by domain-level polymerase chain reaction–denaturing gradient gel electrophoresis (PCR–DGGE). Molecular analysis of enrichment cultures obtained using different ammonia concentrations revealed biases towards Nitrosospira sequence cluster 3 or Nitrosospira sequence cluster 4 under high- or low-ammonia conditions respectively. High-ammonia MPNs suggested a decease in ammonia oxidizer numbers with succession, but low-ammonia MPNs and competitive PCR targeting amoA failed to support such a trend. Ammonia turnover rate, not specific changes in plant diversity and species composition, is implicated as the major determinant of ammonia oxidizer community structure in successional chalk grassland soils.     

黄土丘陵区不同土地利用的土壤水分灰色关联度

运用灰色关联度分析法,分析了黄土丘陵区的延安燕沟不同土地利用方式下土壤水分的垂直变化和月动态.结果表明:梯田各层土壤水分变化态势相似程度较高,即梯田土壤水分的垂直波动较小;不同土地利用方式下均是表层(0-30cm)和中层(30-100cm)土壤水分的灰关联度较大,即土壤水分的变化发展态势较一致,但由于不同利用方式的影响,土壤水分变化态势的相似程度为梯田〉白羊草〉刺槐〉沙棘,除白羊草地外,其他土地利用方式下表层(0-30cm)与深层(100-200)的灰关联度最小,土壤水分变化差异较大.从各月土壤水分灰关联度来看,沙棘除10月份外,其他各月土壤水分变化态势的相似程度较高;白羊草地正好相反,整个生长季土壤水分的变化波动很大;梯田和刺槐林地居中,但各月土壤水分变化态势的相似程度仍然存在差异,表现为雨季前各月土壤水分变化较为一致,雨季后的9、10月份与4月份相比差异较大.说明不同的土地利用方式对土壤水分的垂直变化和月动态变化均产生不同影响. 态势的相似程度为梯田》白羊草》刺槐》沙棘,除白羊草地外,其他土地利用方式下表层(0-30cm)与深层(100-200)的灰关联度最小,土壤水分变化差异较大.从各月土壤水分灰关联度 看,沙棘除10月份外,其他各月土壤水分变化态势的相似程度较高;白羊草地正好相反,整个生长季土壤水分的变化波动很大;梯田和刺槐林地居中,但各月土壤水分变化态势的相似程度仍然存在差异,表现为雨季前各月土壤水分变化较为一致,雨季后的9、10月份与4月份相比差异较大.说明不同的土地利用方式对土壤水分的垂直变化和月动态变化均产生不同影响. 态势的相似程度为梯田》白羊草》刺槐》沙棘,除白羊草地外,其他土地利用方式下表层(0-30cm)与深层(100-200)的灰关联度最小,土壤水分变化差异较大.从各月土壤水分灰关联度 看,沙棘除10月份外,其     

Predisposition of Aster to Phialophora wilt

The susceptibility of Aster novi-belgii to infection by the vascular wilt pathogen Phialophora asteris varied according to the growing conditions to which the host was subjected before inoculation. Relatively low air temperature, high soil moisture, and soil nutrients rich in potassium or phosphorus were pre-inoculation conditions in which Aster became relatively susceptible. Increased resistance was favoured by growing plants at relatively high air temperatures, low soil moisture, and soil nutrients rich in nitrogen or calcium but low in potassium or phosphorus prior to inoculation.