大丽轮枝菌效应蛋白VdSRP2的功能分析

为明确效应蛋白VdSRP2在大丽轮枝菌Verticillium dahliae中的生物学功能,从大丽轮枝菌落叶型菌株V592中克隆VdSRP2基因并进行生物信息学分析,利用酵母转化酶分泌系统对其信号肽活性进行测定,采用实时荧光定量PCR(real-time fluorescence quantitative PCR,qPCR)技术分析VdSRP2基因在大丽轮枝菌中的表达模式,并以V592菌株为材料获得VdSRP2基因的敲除突变体和过表达体菌株,通过表型分析和致病性测定确定VdSRP2基因的生物学功能。结果显示,VdSRP2基因编码232个氨基酸,含有5个半胱氨酸残基,N-端信号肽具有分泌活性,为真菌的典型效应蛋白;VdSRP2基因主要在大丽轮枝菌菌丝和微菌核中表达,其中经棉花根系诱导培养24 h时表达量最高;与野生型菌株V592相比,VdSRP2基因敲除导致大丽轮枝菌的产孢量和孢子萌发率显著下降,不能形成微菌核,对棉花的致病力明显减弱;但VdSRP2基因敲除不影响大丽轮枝菌的穿透能力;VdSRP2基因在本氏烟和棉花叶片瞬时表达不会诱导细胞死亡。表明VdSRP2是大丽轮枝菌微菌核形成必需...     

A chalcone synthase controls the verticillium disease resistance response in both <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and cotton

Verticillium wilt is a devastating disease caused by the soil-borne fungus Verticillium dahliae that causes severe wilt symptoms in more than 400 plant species, including economically important cotton. However, the molecular mechanism of plant resistance to Verticillium remains unclear. In this study, we identified an Arabidopsis mutant, vsad1 (verticillium sensitive and anthocyanin deficient 1), which showed more serious disease symptoms such as discoloration and chlorosis than wild-type Arabidopsis. vsad1 is a previously identified allele of the transparent testa 4 gene (tt4), which encodes chalcone synthase (CHS), a key enzyme involved in the biosynthesis of flavonoids. Our results showed that VSAD1 expression was induced in response to Verticillium dahliae infection. Overexpression of VSAD1 partially recovered the anthocyanin accumulation phenotype of the vsad1–1 mutant. The concentration of V. dahliae increased and ROS accumulation decreased in the vsad1 mutant after infection with V. dahliae. Knockdown of the homologous gene GhCHS in cotton plants increased their susceptibility to V. dahliae infection. Thus, we conclude that VSAD1 is involved in the regulation of plant resistance to Verticillium wilt.     

大丽轮枝菌侵染抗感棉种的组织学过程观察

 棉花黄萎病是一种极难防治的土传性真菌病害,研究病原菌侵染棉花的组织学过程对致病机理解析和抗病资源利用具有重要意义。本研究利用绿色荧光蛋白标记的大丽轮枝菌系统研究了其对抗病棉种海岛棉7124和三裂棉、感病棉种军棉1号和戴维逊棉的侵染过程。结果表明,大丽轮枝菌对抗/感棉种的初始侵染没有明显差异,接菌5 h后,分生孢子均能吸附在感病和抗病棉种的根表面。但侵染过程存在显著差异,侵染感病棉种中病原菌3~5 d到达皮层,5~7 d达到维管束,随后迅速扩展繁殖,侵染14 d后即完成系统侵染,并开始产生黄萎病症状;而病原菌侵染抗病棉种,5~7 d才侵入皮层,7~10 d到达维管束,14 d后仍无法扩展,病原菌的定殖与发展受到限制,无法形成系统侵染,较少形成黄萎病症状。本研究通过绿色荧光蛋白标记大丽轮枝菌对抗/感棉种的侵染过程研究,为大丽轮枝菌致病机理研究和抗性资源利用提供了强有力的理论依据。     

大丽轮枝菌蛋白激发子PevD1诱导棉花抗病性及作用机理

 PevD1是一种大丽轮枝菌(Verticillium dahliae)分泌蛋白,具有激发烟草过敏反应(HR)和系统获得性抗病(SAR)的功能。为明确蛋白PevD1诱导棉花抗病性及其作用机制,本文利用大肠杆菌表达、纯化的PevD1诱导棉苗植株,检测棉苗对大丽轮枝菌的抗性及免疫应答反应。结果表明,大肠杆菌表达的PevD1重组蛋白不是棉花品种“新陆早42号”的致萎因子,叶片注射8 μg/mL PevD1蛋白诱导3 d后根部接种大丽轮枝菌,15 d后PevD1处理组病害减轻率达35.04%。PevD1能诱导棉花叶片抗性早期信号分子H₂O₂产生和NO积累,维管束细胞壁加厚、木质素和酚类物质的积累。另外,PevD1处理能提高防御酶PAL、POD和PPO活性,提高棉花抗性基因和木质素合成相关基因PAL、C4H1、4CL的转录水平。说明PevD1通过激发棉花免疫系统而提高抗病性,该研究不仅为利用PevD1蛋白激发子控制棉花黄萎病提供了科学依据,同时也为阐明棉花与大丽轮枝菌互作机理提供了理论基础。     

Colonization in cotton plants by a green fluorescent protein labelled strain of Verticillium dahliae

Verticillium wilt of cotton (Gossypium hirsutum) is a widespread and destructive disease caused by the soil-borne fungal pathogen Verticillium dahliae. In this study, a green fluorescent protein (GFP) labelled V. dahliae strain (TV7) was obtained by transforming gfp into defoliating strain V991. Strain TV7 was used to study infection and colonization of wilt resistant cotton cultivar Zhongzhimian KV1 and susceptible cultivar 861 with the aid of confocal laser scanning microscopy. The results showed that initial infection and colonization of V. dahliae in Zhongzhimian KV1 and 861 were similar. Conidia and hyphal colonies formed and penetrated in the root meristematic and elongation zones and in the conjunction of the lateral and main roots. The invaded conidia started to germinate by 2 hpi (hours post-inoculation), penetrated into the root cortex and vascular bundles, eventually colonized in the stem xylem vessels and grew restrictedly in the individual tracheae of both resistant and susceptible cultivars. Moreover, pathogen DNA could be detected by qPCR in roots and stems of both cultivars, but its content in the wilt susceptible cultivar 861 was much higher than that in the wilt resistant cultivar Zhongzhimian KV1. The results indicated that the resistant cultivar has ability to suppress V. dahliae reproduction.     

A useful method for preparing microsclerotial inoculum of Verticillium dahliae

Various methods of inoculating plants with Verticillium dahliae have been described; however, so far, there are no effective preparations of its microsclerotia, a major source of infection under natural field conditions. In this study, we developed a simple and effective method to prepare microsclerotial inoculum of V. dahliae using a cellophane medium that effectively promoted microsclerotia formation. In addition, a sieving process allowed the preparation of inoculum exclusively composed of microsclerotia. Our novel method provides a reproducible and stable inoculum for disease induction by V. dahliae under conditions that approximate natural infections.     

Vegetative compatibility groups inVerticillium dahliae isolates from cotton in Turkey

Verticillium dahliae Kleb. with a complicated genetic diversity is a widely distributed major pathogen resulting in cotton wilt, which causes high economic losses in cotton lint production in the cotton belt of Turkey. A collection of 70 TurkishV. dahliae isolates (68 from wilted cotton plants in 28 districts and two from watermelon plants in two districts) were tested for vegetative compatibility by observing heterokaryon formation among complementary nitrate-nonutilizing (nit) mutants. The mutants were tested against international reference tester isolates and also were paired with one another. Thirty-nine isolates were assigned to vegetative compatibility group (VCG) 2B, 19 to VCG2A and three to VCG4B. One isolate was self-incompatible and eight others could not be assigned to any of the identified VCGs because theirnit mutants showed negative reactions with the tester isolates of four VCGs or theirnit mutants reverted back to the wild type. This is the first report of VCGs inV. dahliae from cotton in Turkey.     

新疆棉花黄萎病的发生现状及其病原菌的分子鉴定与ISSR分析

为掌握新疆主要植棉区棉花黄萎病的发生现状及其病原菌大丽轮枝菌Verticillium dahliae的落叶型菌系分布以及遗传变异情况,于2015年对26个新疆主要植棉区棉花黄萎病的发生情况进行了随机调查,统计新疆大丽轮枝菌的培养性状,利用大丽轮枝菌落叶型特异引物D1/D2、INTD2F/INTD2R与非落叶型特异性引物ND1/ND2、INTNDF/INTNDR对新疆大丽轮枝菌菌系进行互补鉴定,并对部分菌系的遗传变异进行简单序列重复区间(inter simple sequence repeat,ISSR)分析。结果表明:2015年新疆棉花黄萎病发病田比例为54.0%,其中病情指数在10.0以上的发病田与2013年持平,而病情指数在20.0以上的严重发病田比例为10.8%,比2013年增加3.8个百分点;新疆大丽轮枝菌的培养性状以菌核型为主,比例为70.1%,菌丝型与中间型比例分别为13.4%和16.5%;新疆大丽轮枝菌落叶型菌系比例为53.2%,26株菌株的来源地全部检出落叶型菌系;聚类分析结果显示,当遗传相似系数为0.66时,新疆大丽轮枝菌落叶型与非落叶型菌系聚为2个谱系,菌系地理来源、培养性状与大丽轮枝菌的遗传分化无明显相关性。     

The influence of Verticillium dahliae and drought on potato crop growth. 1. effects on gas exchange and stomatal behaviour of individual leaves and crop canopies

Potato (Solanum tuberosum L.) plants cv.Saturna were subjected to infection withVerticillium dahliae and drought stress. At the early stages of growth, stomatal conductance, transpiration and net photosynthesis were measured at light saturation (PAR>300 m^–2) on individual leaves and with mobile field equipment with the aid of field enclosures. No significant changes in stomatal conductance and gas exchange characteristics occurred as a result ofV. dahliae instomatal conductance, transpiration and and photosynthetic rates, especially on older leaves and on plants exposed to direct sunlight for a longer period of time. In combination with drought,V. dahliae only occasionally showed interaction; their effects being less than additive. High values of coefficients of variatoon necessitated a high number of measurements per treatment; the more so in the inoculated plants which shows thatV. dahliae seems to affect certain leaves while not affecting others early in growth. Crop photosynthesis was less reduced byV. dahliae than individual leaf photosynthesis due to the levelling effect of integration over the whole canopy and possibly through a stimulation of the top leaves. The upper non-affected leaves are responsible for the bulk of photosynthetic crop activity. The results indicate that following an infection withV. dahliae photosynthesis is reduced early in growth as a result of drought stress in the leaves.Samenvatting Aardappelplanten (Solanum tuberosum L.) cv.Saturna werden onderworpen aan stress als gevolg vanVerticillium dahliae en droogte. In vroege stadia van de groei werden stomataire geleiding, transpiratie en netto fotosynthese bij lichtverzadiging (PAR>300 W m^–2) gemeten aan individuele bladeren en met een mobiel instrumentarium met behulp van gewaskappen. Er werden geen significante verschillen gevonden in de waarden van de stomataire geleiding en de gasuitwisslingskarakteristieken als gevolg vanV. dahliae-besmetting tot een maand na opkomst. Daarna leidde infectie metV. dahliae tot een afname van de stomataire geleiding, transpiratie en netto fotosynthese, speciaal bij oudere bladeren en bij planten die meer aan zonlicht waren blootgesteld. Soms vertoondeV. dahliae interactie met droogte en bleken beide effecten minder dan optelbaar. De hoge waarden van de variatiecoëfficiënten maakten een groot aantal metingen per behandeling noodzakelijk; dit was vooral het geval bij metV. dahliae geïnfecteerde planten hetgeen aantoont datV. dahliae vooral in het begin van de groei niet alle bladeren in gelijke mate aantast. Door de matigende invloed van de integratie van alle bladlagen en mogelijk doordat de bovenste bladeren werden gestimuleerd, werd de totale gewasfotosynthese in mindere mate beïnvloed doorV. dahliae dan de individuele bladfotosynthese. De bovenste niet geïnfecteerde bladeren bleken verantwoordelijk voor het grootste gedeelte van de gewas-fotosynthese. De resultaten tonen aan, dat volgend op een infectie metV. dahliae, de fotosynthese reeds in een vroeg stadium van de groei wordt verminderd als een gevolg van droogtestress in de bladeren.     

Overexpression of StRbohA in Arabidopsis thaliana enhances defence responses against Verticillium dahliae

Plant NADPH oxidases are key regulators of plant–microbe interactions and reactive oxygen species (ROS) are essential to plant defences against pathogens. A significant part in the role played by ROS has been ascribed to plant respiratory burst oxidase homologs (RBOHs). In potato (Solanum tuberosum), where RBOHs were previously shown to be involved in wound-induced oxidative burst, we assessed their expression after inoculation with Verticillium dahliae Kleb. and showed that StRbohA was the only homolog to be differentially induced in potato in response to inoculation. In order to investigate the potential role of this gene in plant protection against wilt diseases, we used Agrobacterium-mediated transformation of Arabidopsis to assess the effects of its overexpression on plant responses to V. dahliae. After inoculation with this pathogen, the transformed Arabidopsis line overexpressing StRbohA showed lower disease severity (percent damaged leaf area and vascular discoloration) as compared to the wild type. It also had higher ROS production and more cell death caused by hydrogen peroxide (H₂O₂), compared to the wild type. Suberization of root cells was also more pronounced in the line overexpressing StRbohA, and supports a possible role for StRBOHA in plant resistance to V. dahliae. Together, these findings indicate that overexpressed StRbohA in Arabidopsis enhances the ROS-mediated defence mechanisms against V. dahliae and can be a potential tool to improve plant resistance to this and other soilborne pathogens that cause wilts in economically important crops.     

Evaluation of Olive Cultivars for Resistance to Verticillium dahliae

Resistance of 23 important olive cultivars to Verticillium dahliae has been evaluated in four experiments under controlled conditions. Nine-month-old nursery olive plants were inoculated with a cotton non-defoliating (ND) (V4) or a cotton defoliating (D) (V117) isolate of V. dahliae. Resistance was evaluated by assessing symptom severity using a 0–4 rating scale and estimating the area under disease progress curves. The percentage of plants killed and of those which recovered from the disease were used as additional parameters for including a particular cultivar into a defined category. Most of the evaluated cultivars were susceptible, although at different levels, to both isolates of V. dahliae. All cultivars were more susceptible to the D pathotype than to the ND one. A group of 11 cultivars, including several important Spanish cultivars, were susceptible or extremely susceptible to both pathotypes of V. dahliae. A second group showed differences of resistance depending on the pathotype used. They were susceptible or extremely susceptible to the D pathotype but resistant or moderately susceptible to the ND one. Finally, 'Frantoio', 'Oblonga' and 'Empeltre' were moderately susceptible to the D isolate of V. dahliae and resistant to the ND one. The resistance of 'Empeltre' was evident by the plant ability to recover from infection with either isolates. 'Empeltre' is considered to be a valuable cultivar for inclusion in breeding programmes for resistance to Verticillium wilt.     

Distribution and persistence of <Emphasis Type="Italic">Verticillium dahliae</Emphasis> in the xylem of Norway maple and European ash trees

Verticillium dahliae colonizes the xylem vessels of susceptible host plants. Hence it can be expected that the distribution of the fungus as well as disease progress will be influenced by the anatomy of the xylem of that host. Here, we studied the spatial and temporal distribution of V. dahliae in relation to recovery from disease symptoms in young European ash (Fraxinus excelsior) and Norway maple (Acer platanoides) trees that differ in vascular anatomy. Quantifying the amount of V. dahliae DNA at different heights in the stem of inoculated trees at different time points after inoculation showed that, in the year of inoculation, the speed of colonization of these two species by V. dahliae was similar. Nevertheless, in the year after inoculation disease incidence and also quantities of V. dahliae detected in maple trees were significantly higher than in ash trees, suggesting that the xylem of ash trees is much less supportive for growth and survival of V. dahliae than that of maple trees. Moreover, in this second year V. dahliae could not be re-isolated from the wood of ash trees that had recovered from disease and was only rarely detected by PCR, only in xylem of the previous year and never in the current xylem. In contrast, V. dahliae easily was detected in the wood of diseased ash and maple trees. Furthermore, despite the presence of a layer of parenchyma cells between growth rings in ash trees, in symptomatic ash trees V. dahliae was present in the xylem of the new growth ring. We also observed that V. dahliae can move downward from the point of inoculation into the root collar, which possibly provides a way for infection of new growth rings by circumventing the physical barriers within the stem xylem.     

Vegetative compatibility groups ofVerticillium dahliae from cotton in the southeastern anatolia region of Turkey

Eighty isolates ofVerticillium dahliae from the southeastern Anatolia region and 20 isolates from the east Mediterranean region from wilted cotton plants were used for vegetative compatibility analysis employing nitrate non-utilizing mutants and reference tester strains of vegetative compatibility groups (VCGs) 1A, 2A, 2B, 3, 4A and 4B. Of the 100V. dahliae isolates, 49 were assigned to VCG1A, 39 to VCG2B, nine to VCG2A and three to VCG4B. Pathogenicity assays were conducted on susceptible cotton cv. Çukurova 1518 in the greenhouse. All VCG1A isolates induced defoliation and all VCG2B isolates caused partial defoliation symptoms. Isolates of VCG2A and VCG4B caused typical symptoms of leaf chlorosis without defoliation. This is the first report on VCGs ofV. dahliae in the southeastern Anatolia region of Turkey, which demonstrates that VCG1A of the cotton-defoliating type and VCG2B of the partially defoliating type are prevalent in this region.     

Weed hosts of <Emphasis Type="Italic">Verticillium dahliae</Emphasis> in cotton fields in Turkey and characterization of <Emphasis Type="Italic">V. dahliae</Emphasis> isolates from weeds

A weed survey conducted in 2004 and 2005 in Aydin province of Turkey showed that Solanum nigrum, Xanthium strumarium, Amaranthus retroflexus, Portulaca oleracea, Sonchus oleraceus and Datura stramonium were the most prevalent weeds in the cotton fields exhibiting Verticillium wilt. Verticillium dahliae Kleb. was recovered from A. retroflexus and X. strumarium in those cotton fields. This is the first report of V. dahliae occurring naturally in A. retroflexus in Turkey. Pathogenicity tests on cotton and weeds showed that the virulence of V. dahliae isolates from weeds was higher on cotton plants than on weeds, with the disease severity ranging from 31.7% to 98.0%. Disease severity of V. dahliae isolates was 54.7–93.9% on eggplant, 23.7–51.6% on cucumber and 11.0–16.4% on tomato, whereas it did not cause any disease symptoms, or only low levels, on pepper and bell pepper. Two vegetative compatibility groups (VCGs) were identified among seven tested weed isolates: VCG2A (two isolates) and VCG2B (three isolates) using international reference strains.     

不同植棉省区落叶型黄萎病菌的培养特性及致病力比较

近年来落叶型症状的棉花黄萎病对棉花生产的危害越来越大,为明确落叶型棉花黄萎病菌的分化特点,采用特异性分子标记方法,对我国10个主要植棉省区30个落叶型棉花黄萎病菌的培养特性和致病力进行了比较。落叶型黄萎病菌系在我国主要植棉省区已普遍存在,该菌系以菌核型为主,其微菌核呈放射状或环状。63.3%菌系的菌丝发达致密,其余菌系的菌丝较为疏松。落叶型黄萎病菌系之间的产孢量和致病力差异较大,产孢量变幅为3.7×107～18.8×107个孢子/mL;71.9%为强致病力菌系,25.0%为中等致病力。研究表明,不同植棉省区间落叶型黄萎病菌系的产孢量和致病力具有一定差异。     

Involvement of two different types of Verticillium dahliae in lettuce wilt in Ibaraki Prefecture, Japan

Although wilt diseases of various plants by Verticillium dahliae occur worldwide, Verticillium wilt of lettuce is relatively new and rare. In 2009, this disease appeared suddenly in commercial fields in Ibaraki Prefecture, Japan. To investigate the cause of the disease, we characterized each lettuce isolate for pathogenicity, mating types, and genetic characteristics and found two types of V. dahliae. A population that included at least two types of V. dahliae was the putative infection source of the disease. Effective management systems against V. dahliae isolates of different races and having different host ranges are necessary.     

Effect of temperature on conidial germination, mycelial growth and aggressiveness of the defoliating and nondefoliating pathotypes of Verticillium dahliae from cotton in China

Three defoliating (D) pathotype isolates and five nondefoliating (ND) pathotype isolates of Verticillium dahliae from cotton in central China were tested for adaptation to various temperatures in conidial germination on water agar (10?C33°C), mycelial growth on potato dextrose agar (10?C33°C) and infection of cotton seedlings (Gossypium hirsutum cvs. ??E Mian 24?? and ??Yin Rui 361??) (25?C33°C). Results showed that the D-pathotype isolates adapted better than the ND-pathotype isolates to 30°C for conidial germination and mycelial growth, although the isolates of the two pathotypes had the same optimum temperature of 25°C. Under day/night temperatures of 25/25, 27/27 and 30/25°C for 20 and 25?days, the D-pathotype isolates induced the defoliation syndrome on seedlings of the two upland cotton cultivars, whereas the ND-pathotype isolates did not induce defoliation syndrome. The values for the areas under the disease progress curve (AUDPC) and the vascular discoloration index (VDI) were higher for the D-pathotype isolates than those for the ND-pathotype isolates in each temperature treatment. Under 30/30 or 33/27°C, at least two of the three D-pathotype isolates still had higher AUDPC values and/or VDI values than all the ND-pathotype isolates on E Mian 24. Therefore, the D-pathotype isolates appear more aggressive than the ND-pathotype isolates in infection of cotton. Results also showed that the cotton cultivar Yin Rui 361 was more tolerant than the cotton cultivar E Mian 24 to infection by both pathotypes of V. dahliae. This study suggests that the D-pathotype isolates can well adapt to high temperature and heavily infect cotton under 25?C30°C, and these features might be responsible for the rapid spread of this pathotype in central China.     

棉花内生真菌CEF-373菌株对棉花黄萎病的防效及其作用机理

为明确棉花内生真菌CEF-373菌株对棉花黄萎病的防效及其作用机理,利用圆盘滤膜法和平板对扣培养法测定菌株CEF-373对大丽轮枝菌Verticillium dahliae菌丝生长的抑制作用,测定其对棉花黄萎病的温室和田间防效,并通过活性氧含量及防御基因表达情况来分析其作用机理。结果表明,菌株CEF-373的挥发性代谢产物和非挥发性代谢产物对大丽轮枝菌菌丝的生长均有显著抑制作用,抑制率最高分别可达37.75%和100.00%。用1×107CFU/mL的菌株CEF-373分生孢子悬浮液灌根后,对棉花黄萎病的温室防效可达71.12%,用质量比为3%的菌株CEF-373固体菌剂拌土栽培后,对棉花黄萎病的温室防效可达62.74%,防治作用显著。菌株CEF-373的发酵液滴灌和微生物肥料处理40 d后,对棉花黄萎病的田间防效达到最大,分别为36.23%和27.71%,而在后期有所降低。菌株CEF-373可以诱导棉花叶片中细胞活性氧的爆发;且菌株CEF-373成功激活了苯丙氨酸解氨酶、过氧化物酶、多酚氧化酶、几丁质酶和病程相关蛋白基因PR10的表达,对大丽轮枝菌的侵染具有抵御作用。表明棉花内生真菌CEF-373菌株通过抑制大丽轮枝菌生长以及诱导寄主系统抗病性来有效防治棉花黄萎病,具有较好的生物防治应用前景。     

Reliable detection of unevenly distributed Verticillium dahliae in diseased olive trees

Verticillium wilt caused by Verticillium dahliae is one of the most threatening diseases of olive worldwide. For pre‐planting and post‐planting control of verticillium wilt in olive trees, availability of a rapid, reliable and non‐destructive method for detection of V. dahliae is essential. For such a method, suitable and easily performed sampling and efficient processing of samples for extraction of DNA are necessary. In this study, the suitability of young twig and leaf samples of olive trees, which are easy to collect and extract DNA from, were assessed for the detection of V. dahliae in routine procedures. The lower (about 50 cm from the tip) and top parts (about 5 cm from the tip) of twigs, as well as leaves from infected olive trees were screened for V. dahliae infection and distribution using real‐time PCR. The biomass of V. dahliae detected in individual twigs was highly variable, but there was no significant difference between mean quantities of V. dahliae DNA detected in top and lower parts of twigs. Furthermore, it was demonstrated that analysis of combined samples containing DNA extracted from five twigs of an infected tree accurately detected the presence of the pathogen. Similarly, testing combined samples of 5–10 leaves enabled reliable detection of the pathogen in an infected tree. The development of this assay enables reliable detection of V. dahliae in infected olive trees that can aid in management decisions for the implementation of integrated disease management.