Characterization of <Emphasis Type="Italic">Phytophthora clandestina</Emphasis> races on <Emphasis Type="Italic">Trifolium subterraneum</Emphasis> in Western Australia

Phytophthora clandestina is a causal agent of root rot disease of subterranean clover in Western Australia (W.A). As a significant number of isolates of P. clandestina from W.A. could not previously be designated using existing differentials, a comprehensive set of subterranean clover (Trifolium subterraneum) cultivars was used as differentials to delineate a broader range of races of the pathogen. One hundred and one isolates of the pathogen collected from W.A. were screened on nine subterranean clover cultivars, of which seven were found to be useful as host differentials. A total of 10 races (in contrast to the five recognized previously) were defined and differentiated using octal nomenclature, presenting a clearer picture of the racial distribution of P. clandestina among W.A. isolates. Differences were found in the race populations between Australian states and are therefore important to the selection/breeding of cultivars for specific regions of Australia to counter the predominant race populations and for enforcing quarantine measures in relation to seed movements within and outside Australia. The octal nomenclature used provides a sound basis for follow-up studies and future race designations. Races 173 and 177 in this study were widely distributed and were the most common races in W.A., and together constitute 80% of the isolates characterized. While six of the seven host differentials were resistant to isolates belonging to race 001 and all were resistant to 000, it is of concern that only one differential was resistant to 157 and 173 and that none of the host differentials were resistant to 177. Our approach to P. clandestina race delineation is clearly conservative and is different from previous studies. The octal nomenclature we applied in this study is not only scientifically sound but also will facilitate rapid recognition and characterization of the races.     

Development of a Real-Time Polymerase Chain Reaction Assay for Quantifying Verticillium albo-atrum DNA in Resistant and Susceptible Alfalfa

ABSTRACT A precise real-time polymerase chain reaction (PCR) assay was developed for quantifying Verticillium albo-atrum DNA. The assay was used in a repeated experiment to examine the relationship between the quantity of pathogen DNA detected in infected leaves and shoots and the severity of Verticillium wilt symptoms in several alfalfa cultivars expressing a range of disease symptoms. Plants were visually inspected for symptoms and rated using a disease severity index ranging from 1 to 5, and the quantity of pathogen DNA present in leaves and stems was determined with real-time PCR. No significant differences in pathogen DNA quantity or disease severity index were observed for experiments or for cultivar-experiment interactions. Significant differences were observed between cultivars for the quantity of pathogen DNA detected with real-time PCR and also for disease severity index ratings. In both experiments, the highly resistant check cultivar Oneida VR had significantly less pathogen DNA, and significantly lower disease severity index ratings than the resistant cultivar Samauri, the moderately resistant cultivar Vernema, and the susceptible check cultivar Saranac. In both experiments, the Spearman rank correlation between the amount of V. albo-atrum DNA detected in leaves and stems with real-time PCR and disease severity index ratings based on visual examination of symptoms was positive (>0.52) and significant (P < 0.0001). These results suggest that resistance to Verticillium wilt in alfalfa is characterized by a reduced colonization of resistant genotypes by the fungus.     

Resistance in water yam (Dioscorea alata) cultivars in the French West Indies to anthracnose disease based on tissue culture-derived whole-plant assay

Reactions of 60 water yam ( Dioscorea alata ) cultivars to three isolates of the yam anthracnose fungal pathogen ( Colletotrichum gloeosporioides ) were evaluated using tissue culture-derived whole-plant assay. Three disease parameters: single score on a scale of 0–6 at the seventh day after inoculation (SD7); area under the disease progress curve (AUDPC); and disease progress rate ( R _d) were compared, and cultivars were classified into disease-response groups using a rank-sum method based on AUDPC scores for the two most virulent isolates. A wide range of variation in resistance of the D. alata cultivars, and significant effects of pathogen isolate and isolate–cultivar interactions, were observed for all disease parameters. The three disease parameters were positively correlated; however, four cultivars showed great dispersions from the regression lines for comparisons of SD7 with the multiple assessments based AUDPC and R _d. The 60 cultivars were separated into resistant ( n  = 12), moderately resistant ( n  = 19), moderately susceptible ( n  = 18) and susceptible ( n  = 11) groups. The potential of the tissue culture-derived whole-plant assay to resistance breeding programmes and further understanding of the yam anthracnose pathosystem is discussed.     

脱氧雪腐镰刀菌烯醇在小麦赤霉病病程中的作用

 脱氧雪腐镰刀菌烯醇(deoxynivalenol,DON)系小麦赤霉病菌——禾谷镰孢(Fusarium graminearum,有性态Gibberell azeae)产生的一种单端孢霉烯族毒素(trichothecenes,以下简称"单族毒素")。作者以病菌正常产毒的野生型菌株GZ3639及其产毒基因Tri5损坏、不能产毒的转化子GZT40(Tri5^-,TRI^-)为材料,对DON在病菌致病过程中的作用进行了研究。结果显示:GZ3639能引起典型的赤霉病症状,病害可由接种小花发展至其它小穗,病菌也可定殖于其它小穗组织;而GZT40仅在接种小花处产生鸟眼状坏死斑点,不能引起典型的赤霉病症状,病菌仅定殖于接种小花处。当病菌与一定浓度(400 μ g·mL^-1)的DON混合处理小花后,GZT40的定殖时间提前2 d (感病品种上)至4 d (抗病品种上),定殖范围可扩大到1~3个小穗,接种20 d后的病小穗率也由病菌单独接种时的2.8%提高到12.5%。DON也使GZ3639的定殖时间提前1 d (感病品种上)至2 d (抗病品种上),定殖范围更广,其所致的病害发展更迅速,接种后20 d的病小穗率由病菌单独接种时的78.0%提高到100%。因此,作者认为,单族毒素在病害发展和病菌在寄主组织内的扩展中起着决定性作用,一旦丧失产毒能力,病菌虽能侵入寄主,但不能在寄主组织中扩展。DON可以缩短病菌成功定殖寄主所需的时间,更有助于病菌在寄主组织内的扩展。     

Structure and development of decision‐support systems and their use by the State Plant Protection Services in Germany*

To ensure continuing work on decision‐support systems (DSSs) and the elaboration of new systems according to the needs of the Plant Protection Services of the federal states of Germany, these services have installed ZEPP (Central Institution for Decision Support Systems and Programmes in Crop Protection). At the national level, ZEPP guarantees a permanent supply of meteorological data, organizes and co‐ordinates trials, incorporates scientific progress into the existing DSSs and, in close co‐operation with universities and federal research stations, develops new systems for important pests. The PASO desktop system includes a data bank that administers all relevant meteorological files and field data. It allows for automated simulation runs and forecasts, serving as input for timely warnings by different media. Depending on the individual decision‐support modules, results are presented as tables and/or graphs.     

Somaclonal selection in potato for resistance to common scab provides concurrent resistance to powdery scab

Variant somaclones of potato cultivar Russet Burbank, selected for resistance to common scab using in vitro cell selection techniques, were tested for resistance to powdery scab, another important disease of potato caused by Spongospora subterranea. This pathogen also invades roots, producing root galls. Most variants consistently showed increased resistance to powdery scab, both in field and glasshouse challenge, when compared to the parental cultivar, several significantly so. On average, the best variant reduced powdery scab incidence by 51% and severity (tuber surface coverage) by 64%. In contrast, no improvement in the extent of root infection and root galling was seen. These results suggest host interactions during root and tuber infection are distinct. Correlation analyses of disease indices amongst the selected variants showed no association between Sp. subterranea root infection and gall scores, nor between root infection and tuber disease severity. However, a weak positive association was found between root gall score and tuber disease, and a strong correlation between tuber disease incidence and severity scores. Interestingly, positive correlations were also found between the extent of powdery and common scab resistance expressed and both incidence and severity of these diseases within the variants, suggesting a common defence mechanism. The role of thaxtomin A in selecting for concurrent resistance to both diseases is discussed.     

Durability of Resistance and Cost of Virulence

A seasonal model, where a growing season is defined as the time between sowing and harvest and alternates with an inter-crop period, was derived to study the effects of the ‘cost of virulence’ and cropping ratio on durability of resistance. We assumed a single strain of virulent pathogen, a single strain of avirulent pathogen and two cultivars (one resistant and one susceptible) and studied two measures of durability of resistance (‘take-over time’ and ‘usefulness time’). Take-over time is defined as the time needed for the virulent strain of the pathogen to reach a preset threshold and predominate over the previous pathogen population. Usefulness time is the time needed before the estimated gain in green canopy area duration per plant through the use of the resistant cultivar becomes negligible. The model suggested that, although it could take several seasons before the virulent strain of the pathogen predominated over the previous pathogen population, the usefulness time of the resistant cultivar was always much shorter. Furthermore, increasing selection for the virulent strain of the pathogen (through increasing the cropping ratio of the resistant cultivar) caused the virulent strain of the pathogen to invade the system more rapidly. Cost of virulence, reflecting differences in pathogen infection rates between the four possible combinations of cultivar/pathogen strain, significantly affected durability of resistance, with the dynamics of the virulent and avirulent strains ranging from a case where the virulent strain of the pathogen died out to a case where the virulent strain of the pathogen invaded the resident pathogen population. An intermediate state, where the system reached equilibrium and the virulent strain of the pathogen neither became predominant nor died out, was defined as ‘coexistence’ of both strains of the pathogen. Occurrence of coexistence was directly related to the cost of virulence since it did not occur when virulence of the pathogen did not have a fitness cost. Two methods to include cost of virulence in the model gave similar results in relation to the two measures of durability of resistance studied.     

新疆野苹果和秦冠的抗黑星病特性

 透射电镜观察表明, 苹果叶片上表皮角质层厚度在品种间存在显著差异, 新疆野苹果和秦冠叶片的角质层厚度显著高于富士和嘎啦的;同一品种不同龄期叶片的角质层厚度随着叶龄增长而增厚, 且黑星病严重度与叶片上表皮角质层厚度间存在显著的负相关关系。苹果品种抗病性组分分析结果表明, 新疆野苹果的病害严重度最低, 约为嘎啦的1/22, 潜育期最长, 为嘎啦的2.0倍, 无(或少有)病斑出现, 不产孢。秦冠的严重度约为嘎啦的1/14, 潜育期约为嘎啦的1.5倍, 产孢量约为嘎啦的1/26。黑星病菌在新疆野苹果和秦冠叶片上的侵染概率及病斑扩展速率均显著低于富士和嘎啦。因此, 新疆野苹果和秦冠对黑星病的抗病性表现在抗侵入和抗扩展两个方面。     

黄瓜根分泌物中的7,8-苯并黄酮及其对枯萎病发生的影响

分别提取黄瓜枯萎病感病品种津研4号和抗病品种中农10号的根分泌物,感病品种根分泌物甲醇提取液能够刺激枯萎病菌的菌丝生长,而抗病品种根分泌物甲醇提取液则抑制病原菌生长。利用LC-MS/MS技术分别对感病品种和抗病品种根分泌物中的差异物质进行分析鉴定。与感病品种津研4号相比,抗病品种中农10号根分泌物中含有7,8-苯并黄酮(7,8-BF),含量为0.02μg/株。50μg/mL的7,8-BF能够显著抑制黄瓜枯萎病菌菌丝生长和孢子萌发,而100μg/mL的7,8-BF能显著抑制抗感品种侧根形成,但是抗病品种的耐受力高于感病品种。用7,8-BF处理黄瓜种子,枯萎病侵染程度显著降低。当7,8-苯并黄酮浓度为50μg/mL时,感病品种病情指数从71.7降为30.8,达到中抗水平。     

Improved control of septoria tritici blotch in durum wheat using cultivar mixtures

Mixtures of cultivars with contrasting levels of resistance can suppress infectious diseases in wheat, as demonstrated in numerous field experiments. Most studies focus on airborne pathogens in bread wheat, while splash-dispersed pathogens have received less attention, and no studies have been conducted in durum wheat. We conducted a 2-year field experiment in Tunisia to evaluate the performance of cultivar mixtures with varying proportions of resistance (0%–100%) in controlling the polycyclic, splash-dispersed disease septoria tritici blotch (STB) in durum wheat. To measure STB severity, we used a high-throughput method based on digital image analysis of 3,074 infected leaves collected from 42 and 40 experimental plots during the first and second years, respectively. This allowed us to quantify pathogen reproduction on wheat leaves and to acquire a large data set that exceeds previous studies with respect to accuracy and precision. Our analyses show that introducing only 25% of a disease-resistant cultivar into a pure stand of a susceptible cultivar provides a substantial reduction of almost 50% in disease severity compared to the susceptible pure stand. However, incorporating two resistant cultivars instead of one did not further improve disease control, contrary to predictions of epidemiological theory. Susceptible cultivars can be agronomically superior to resistant cultivars or be better accepted by growers for other reasons. Hence, if mixtures with only a moderate proportion of the resistant cultivar provide a similar degree of disease control as resistant pure stands, as our analysis indicates, such mixtures are more likely to be accepted by growers.     

Rating and mapping the suitability of the climate for pest risk analysis*

The assessment of the suitability of the climate for pest establishment is an important part of pest risk analysis (PRA). This paper describes the work undertaken by the EU 7th Framework project PRATIQUE (Enhancements of Pest Risk Analysis Techniques) to develop guidance for this component of PRA. Firstly, there is a guide to rating the suitability of the climate in the PRA area using qualitative methods. Secondly, a Decision‐support scheme (DSS) has been created to assist analysts in deciding whether to map climatic suitability, and to guide the selection of the most appropriate method from the large number available. The process of selecting a climatic mapping method is based on a review of the pest’s climatic responses and distribution. A spreadsheet provides a comparison of the potential problems that can arise, depending on the mapping method and on the amount and quality of available data. Diagrams are provided to help choose the location data category that best represents the possible biases in the known distribution of the pest. A second spreadsheet provides general information on the differences and similarities of each method in terms of categories such as functionality, ease of use and quality assurance. A variety of data, tools and supporting documents are available as appendices to the DSS. All of the tools and guides are freely available online.     

Effect of temperature on the incubation period and leaf colonization in bacterial blight of anthurium

ABSTRACT Effect of temperature on leaf colonization in anthurium blight was studied using a bioluminescent strain of Xanthomonas campestris pv. dieffenbachiae. In a susceptible cultivar, colonization of leaf tissues (monitored by detection of bioluminescence) and symptom development (assessed visually) advanced rapidly at higher temperatures. For a susceptible cultivar, there was a linear relationship between degree-days and percent leaf area colonized by the pathogen, indicating that leaf colonization in a susceptible cultivar was a direct function of the cumulative effect of temperature. The degree-day intercept of the regression line represented the time from inoculation to detection of bioluminescence, and the slope indicated the increase of leaf colonization per degree-day. There also was a linear relationship between the logarithm of degree-days and the logarithm of percent leaf area showing visible symptoms in a susceptible cultivar. The degree-day intercept of this relationship represented the incubation period (about 500 degree-days). The degree-days required to detect bioluminescence was not considerably different between susceptible and resistant cultivars. However, the subsequent rates of leaf colonization were significantly lower for a resistant cultivar than for a susceptible cultivar in all temperature regimes. The results suggest that multiplication of the pathogen in the leaf tissues is optimized in the susceptible cultivar. In contrast, in the resistant cultivar, the defense mechanisms overshadow the temperature effect. The differential response to temperatures may be an additional indicator of cultivar susceptibility.     

Pathotypes and phylogenetic variation determine downy mildew epidemics in Brassica spp. in Australia

Isolates of Hyaloperonospora brassicae inoculated onto cotyledons of 28 diverse Brassicaceae genotypes, 13 from Brassica napus, two from B. juncea, five from B. oleracea, two from Eruca vesicaria, and one each from B. nigra, B. carinata, B. rapa, Crambe abyssinica, Raphanus sativus and R. raphanistrum, showed significant effects (P ≤ 0.001) of isolate, host and their interaction. Host responses ranged from no visible symptom or a hypersensitive response, to systemic spread and abundant pathogen sporulation. Isolates were generally most virulent on their host of origin. Using an octal classification, six host genotypes were identified as suitable host differentials to characterize pathotypes of H. brassicae and distinguished eight distinct pathotypes. There were fewer, but more virulent, pathotypes in 2015–2016 isolates than 2006–2008 pathogen populations, probably explaining the increase in severity of canola downy mildew over the past decade. Phylogenetic relationships determined across 20 H. brassicae isolates collected in 2006–2008 and 88 isolates collected in 2015–2016 showed seven distinct clades, with 70% of 2006–2008 isolates distributed within clade I (bootstrap value (BVs) of 100%) and the remaining 30% in clade V (BVs 83.3%). This is the first study to define phylogenetic relationships of H. brassicae isolates in Australia, setting a benchmark for understanding current and future genetic shifts within pathogen populations; it is also the first to use octal classification to characterize pathotypes of H. brassicae, providing a novel basis for standardizing phenotypic characterization and monitoring of pathotypes on B. napus and some crucifer species in Australia.     

Virulence phenotypes in powdery mildew (Blumeria graminis) populations and resistance genes in triticale (x Triticosecale)

Triticale (xTriticosecale) is a new host for powdery mildew (Blumeria graminis) being not infected by this pathogen in Germany before 2001. To evaluate population structure of the pathogen and race-specific resistances in the host, 694 isolates were collected in 12 states of Germany in the years 2007 to 2009 on triticale. They were tested by a newly developed initial differential set of 20 triticale cultivars. Corresponding virulences were found for all differentials except for cultivar Grenado. In total, 272 different virulence phenotypes (=pathotypes) were detected. The virulence complexity of the isolates ranged from 6 to 19 of 20 possible virulences with a mean of 15. In all years, a high level of diversity of the powdery mildew populations was observed with Simpson indices in the range 0.95 to 0.97. The distribution of the pathotypes was even across Germany with an Evenness index in the range 0.82 to 0.88. A set of 19 isolates with different virulence pathotypes and 10 cultivars were selected to be used to identify race-specific resistances of triticale cultivars and breeding lines. Some cultivars susceptible to most of the isolates in seedling stage were moderately resistant in adult-plant stage. The high diversity and complexity of the pathotypes found in German powdery mildew populations as well as an increasing acreage of only a few dominating triticale cultivars accelerate the adaptation of the pathogen to race-specific host resistances suggesting restricted durability only. More durable resistance might be achieved by combining new effective race-specific (qualitative) resistance genes with race-nonspecific (quantitative) resistances effective in the adult-plant stage that are already available.     

品种布局防治小麦条锈病的模拟研究

 用小麦条锈病大区流行模型PANCRIN进行的模拟试验结果指出:抗病品种的合理布局能有效地延缓和减轻抗病性丧失,但其效果易受品种面积、流行速率和越夏区面积浮动的影响。布局策略上首先要按照小麦条锈病菌越夏途径中不同成熟期的麦田进行细致的布局,也就是布局要和"打越夏"结合,效果才更显著。其次要控制每个抗病品种的面积而增多抗病品种的数目,在所用模型规定的条件下,每一抗病品种的种植面积百分比不可超过15%,抗病品种总数争取≥ 6个。此外,要尽量压缩感病品种面积,减少大区流行菌量,感病品种面积最好不超过10%。流行系统的定量控制是个极其复杂的问题,因此上述初步结果只能供作研究参考,还不能视为应用指标。合理布局的设计应以深入掌握越夏规律为基础,这需要进一步开展有关研究。目前情况下,生产上尽量增多抗病品种数目,只要上下游品种雷同的不多,也能多少延长抗病品种的使用年限。     

Fusion of sensor data for the detection and differentiation of plant diseases in cucumber

The development of plant diseases is associated with biophysical and biochemical changes in host plants. Various sensor methods have been used and assessed as alternative diagnostic tools under greenhouse conditions. Changes in photosynthetic activity, spectral reflectance and transpiration rate of diseased leaves, inoculated with Cucumber mosaic virus (CMV), Cucumber green mottle mosaic virus (CGMMV), and the powdery mildew fungus Sphaerotheca fuliginea were assessed by the use of non‐invasive sensors during disease development. Spatiotemporal changes in leaf temperature related to transpiration were visualized by digital infrared thermography. The maximum temperature difference within a leaf was an appropriate parameter to differentiate between healthy and diseased plants. The photosynthetic activity of healthy and diseased cucumber plants varied as measured by chlorophyll fluorescence and compared to the actual chlorophyll content. Hyperspectral imaging data were analysed using spectral vegetation indices. The results from this study confirm that each pathogen has a characteristic influence on the physiology and vitality of cucumber plants, which can be measured by a combination of non‐invasive sensors. Whereas thermography and chlorophyll fluorescence are unspecific indicators for plant diseases, hyperspectral imaging offers the potential for an identification of plant diseases. In a sensor data fusion approach, an early detection of each pathogen was possible by discriminant analysis. Although it still needs to be validated under real conditions, the combination of information from different sensors seems to be a promising tool.     

Identification of Molecular Genetic Markers in Pyrenophora teres f. teres Associated with Low Virulence on 'Harbin' Barley

ABSTRACT Two isolates of the barley net blotch pathogen (Pyrenophora teres f. teres), one possessing high virulence (0-1) and the other possessing low virulence (15A) on the barley cultivar Harbin, were crossed and the progeny of the mating were isolated. Conidia from cultures of the parent and progeny isolates were used as inoculum to determine the inheritance of virulence in the pathogen. Of the 82 progeny tested, 42 exhibited high virulence and 40 exhibited low virulence on 'Harbin' barley. The data support a model in which a single, major gene controls virulence in P. teres f. teres on this barley cultivar (1:1 ratio; chi(2) = 0.05, P = 0.83). Preparations of DNA were made from parental and progeny isolates, and the DNA was subjected to the random amplified polymorphic DNA (RAPD) technique in a search for molecular genetic markers associated with the virulence phenotype. Five RAPD markers were obtained that were associated in coupling with low virulence. The data indicate that the RAPD technique can be used to tag genetic determinants for virulence in P. teres f. teres.     

Effects of the host, the pathogen, the environment and their interactions, on fusarium wilt in carnation

Fusarium oxysporum f.sp.dianthi causes severe wilting in carnation (Dianthus caryophyllus L.) worldwide. The pathogen is present in the soil profile in which carnation roots are distributed and may infect the plants at any time during the growing season. To minimize the losses induced by Fusarium wilt, growers use carnation cuttings free ofFusarium spp. and fumigate the soil with methyl bromide prior to planting. The severity of epidemics and the resulting losses are governed by the main and interacting effects of the three components of the disease syndrome: the host, the pathogen and the environment. Host variables include the type and the degree of cultivar resistance (i.e., complete, partial or tolerance); pathogen variables include the race, its virulence and infectivity, and the amount of initial inoculum; environmental variables include solar radiation intensity, photoperiod, temperature and the growth substrate. In the present review the information available on the effect of the host, the pathogen and the environment, and their interactions, on Fusarium wilt in carnation is summarized.     

Computerized information system for cereal disease management in Emilia-Romagna (Italy)1

CERMAL is a computerized system on cereal diseases in Emilia-Romagna (IT), holding yearly data on severity of fungal diseases (powdery mildew, rusts, septoria diseases, scab, net and spot blotch, scald and black point) on bread wheat, durum wheat, barley and triticale collected in the experimental trials of the Union of Experimental Stations in Emilia-Romagna (CERAS) since 1979. The data base contains information on approximately 300 experimental trials. Basic trial data (year, location, cereal-growing area) and severity of disease on each cultivar are stored. A management program allows data to be retrieved on the basis of year, cereal-growing area and/or cultivar, for use in statistical analyses. Information on the behaviour of cultivars in relation to disease, the probability of disease appearance and development, and the level of disease risk in a given cereal-growing area can be output and used for cultivar choice and disease management. CERMAL operates within the integrated production program for wheat and barley supported by the Emilia-Romagna region.     

Factors Affecting Pseudomonas savastanoi pv. savastanoi Plant Inoculations and Their Use for Evaluation of Olive Cultivar Susceptibility

ABSTRACT Pseudomonas savastanoi pv. savastanoi causes olive knot disease, which is present in most countries where olive trees are grown. Although the use of cultivars with low susceptibility may be one of the most appropriate methods of disease control, little information is available from inoculation assays, and cultivar susceptibility assessments have been limited to few cultivars. We have evaluated the effects of pathogen virulence, plant age, the dose/response relationship, and the induction of secondary tumors in olive inoculation assays. Most P. savastanoi pv. savastanoi strains evaluated were highly virulent to olive plants, but interactions between cultivars and strains were found. The severity of the disease in a given cultivar was strongly dependent of the pathogen dose applied at the wound sites. Secondary tumors developed in noninoculated wounds following inoculation at another position on the stem, suggesting the migration of the pathogen within olive plants. Proportion and weight of primary knots and the presence of secondary knots were evaluated in 29 olive cultivars inoculated with two pathogen strains at two inoculum doses, allowing us to rate most of the cultivars as having either high, medium, or low susceptibility to olive knot disease. None of the cultivars were immune to the disease.