Pythium violae isolated from cavity spot lesions on carrots in Israel

Pythium violae Chesters & Hickman was isolated for the first time from cavity spot lesions on carrots in Israel and the resulting isolates were compared with isolates from the UK, France and Spain. The isolates from all locations responded similarly to temperature and had similar colony morphology, oogonial diameters and soluble protein patterns.     

Ultrastructural and cytochemical investigations of pathogen development and host reactions in susceptible and partially-resistant carrot roots infected by Pythium violae, the major causal agent for cavity spot

Two carrot genotypes, cultivar Nanco and line 24, susceptible and partially- resistant respectively to cavity spot, were compared ultrastructurally and cytochemically 24 h, 48 h and 72 h after root inoculation with a virulent Pythium violae isolate. The extent of pathogen ingress and the response of the host differed markedly with the two genotypes. In cv Nanco, growth of fungal hyphae was predominantly intracellular and was accompanied by pronounced damage; by 48 h after inoculation, pericycle and the first cell layers of the phloem parenchyma were invaded, resulting in host wall dissolution and cytoplasm aggregation. The growth of P. violae in line 24 was limited to the pericycle, even up to 72 h after inoculation; fungal colonization was accompanied by retraction of cytoplasm and in the appearance of granular or fibrillar material in the host cell lumen. Some affected host cells were filled with structureless osmophilic material. In cultivar Nanco, invading fungal hyphae were unaffected; by contrast in line 24, the cytoplasm of invading hyphae, particularly those inside the cell host, was disorganised and structureless. Infection and host response in the two cultivars were studied with two specific labels: Aplysia gonad lectin (AGL), a polygalacturonic acid-binding lectin, and an exoglucanase complexed to colloidal gold were used to locate pectin and cellulosic -(1,4)-glucans respectively in infected tissues. The decrease of cytochemical labeling beyong fungal penetration showed clearly hydrolysis of pectin and cellulose in cell walls of the cv Nanco. By contrast, the cell wall of line 24 remained largely intact, although, unlabeled amorphous and electron-dense material was observed inside the wall. Fibrillar or electron dense material commonly observed in infected tissue of line 24 apparently did not contain pectic or cellulosic substances. Moreover, material observed in host cells or fungal hyphae was also free of labeling. The origin and the chemical composition of these compounds as well as their possible role in the defence mechanisms of carrot against P. violae are discussed.     

Demonstration of secondary infection by Pythium violae in epidemics of carrot cavity spot using root transplantation as a method of soil infestation

Cavity spot of carrot (CCS), one of the most important soilborne diseases of this crop worldwide, is characterized by small sunken elliptical lesions on the taproot caused by a complex of pathogens belonging to the genus Pythium , notably P. violae . In most soilborne diseases the soil is the source of inoculum for primary infections, with diseased plants then providing inoculum for secondary infections (both auto- and alloinfection). Using fragments of CCS lesions to infest soil, it was demonstrated that CCS lesions on carrot residues can cause primary infection of healthy roots. Using a novel soil infestation method, in which an artificially infected carrot root (the donor plant) was placed close to healthy roots (receptor plants) the formation of typical CCS lesions were induced more efficiently than the use of classical soil inoculum and showed that CCS can spread from root to root by alloinfection from transplanted diseased roots. The method also demonstrated the polycyclic nature of a CCS epidemic caused by P. violae in controlled conditions. Secondary infections caused symptoms and reduced root weight as early as two weeks after transplantation of the diseased carrot. This reproducible method may be used for delayed inoculation and for studying the effect of cropping factors and the efficacy of treatments against primary and secondary cavity spot infections.     

Aggressiveness and production of cell-wall degrading enzymes by Pythium violae, Pythium sulcatum and Pythium ultimum, responsible for cavity spot on carrots

This study investigated the relationships between pathogenesis, types of symptoms and in vitro production of cell-wall degrading enzymes by P. violae, P. sulcatum and P. ultimum. The three pathogens, considered as the three Pythium species principally responsible for cavity spot on carrot roots, secreted only low levels of fatty acid esterases activity, suggesting they have limited ability to degrade suberin in the walls of the outer cell layers of carrot tissues. Among the enzymes that degrade cell-wall polysaccharides, only pectate lyases and cellulases were produced by P. violae, and these were produced late and in small amounts: the symptoms caused by P. violae were limited and typical of cavity spot. Conversely, P. ultimum caused maceration of tissues, and secreted polygalacturonases and -1,4-glucanases earlier and in larger amounts than P. violae. P. ultimum also produced a large diversity of proteins and cellulase isoenzymes. Although secreting all the monitored enzymes in higher quantity than the two previous species, P. sulcatum was responsible for only typical limited symptoms of cavity spot, with a brown colouring. The role of plant reactions induced in response to early pectinolytic enzyme production by P. sulcatum may account for this apparent inconsistency.Died on April 11, 1997     

Modulation of primary and secondary infections in epidemics of carrot cavity spot through agronomic management practices

The relative importance of primary and secondary infections (auto- and alloinfections) in the development of a carrot cavity spot (CCS) epidemic caused by Pythium spp. were investigated. Three cropping factors: fungicide application, soil moisture and planting density, were selected as the key variables affecting the disease tetrahedron. Their effects on: (i) disease measurements at a specific time, (ii) the areas under the disease progress curves (AUDPCs) and (iii) a time-dependent parameter in a pathometric incidence-severity relationship, were studied. Mefenoxam applications 5 and 9 weeks after sowing reduced the intensity of a field CCS epidemic that involved both primary and secondary infections. In microcosm experiments, mefenoxam reduced secondary infections by Pythium violae obtained by transplanting infected carrot roots and slowed disease progress (1·6 lesions per root in treated versus 5·8 lesions in non-treated microcosms). A deficit of soil moisture limited the movement of Pythium propagules to host tissue, and thus reduced primary infections in the field; it also promoted the healing of lesions, limiting lesion expansion and the potential for alloinfections (6·8–7·5 lesions per root in irrigated plots compared with 2·4 lesions in non-irrigated plots). A negative relationship between the mean root-to-root distance and the rate of alloinfections was established in microcosms; a reduction in mean planting density was also effective in limiting CCS development (0·5, 1·6 and 2·0 lesions per root in microcosms containing 8, 16 and 31 roots, respectively). An integrated disease management system based on a combination of cultural methods, such as optimized fungicide application, date of harvest versus soil moisture content, and host density versus planting pattern, may make a useful contribute to the control of CCS.     

Reduced persistence of metalaxyl in soil associated with its failure to control cavity spot of carrots

Metalaxyl was used to control Pythium diseases of carrots in experiments on farms with a history of cavity spot. The first experiment compared the method of application (sprayed, banded or broadcast) and rate (0, 1.5, 3 or 6 kg a.i. ha⁻¹) one week after sowing. Three additional experiments compared the rate (0, 0.75, 1.5 or 3 kg a.i. ha⁻¹) and time (sowing, 1- to 2- or 4- to 5-true-leaf stage) of application. In expt 1, the application of metalaxyl, but not the method by which it was applied, increased yield by 20% and significantly reduced the incidence of cavity spot, forking and misshapen carrots. In expts 2, 3 and 4, neither the rate nor time of application affected yield or reduced the incidence of Pythium diseases. Comparison of the sites showed that they differed in past metalaxyl usage. Metalaxyl had not been used on the site of expt 1, but had been used previously at sites 2, 3 and 4. Laboratory experiments were conducted to determine whether these differences in efficacy resulted from reduced sensitivity of Pythium isolates to metalaxyl, or reduced persistence of metalaxyl in soil. ED₅₀ values showed that there was no reduction in metalaxyl sensitivity. The half-life of metalaxyl was 82 days in soil from expt 1, but was 10 days or fewer in soils from expts 2, 3 and 4. Thus the failure of metalaxyl to control Pythium diseases was associated with reduced persistence in soil, not reduced sensitivity of the target fungi.     

Evaluation of fungicides for the control of carrot cavity spot

Cavity spot is one of the most common and serious diseases of carrot (Daucus carota L). The disease, caused by different species of Pythium, including P. violae Chesters & Hickman, P. sulcatum Pratt & Mitchell and P. sylvaticum Campbell & Hendrix, leads to frequent high rejection rates during grading worldwide. In the area of the city of Québec, the disease is caused mainly by P. sulcatum, P. sylvaticum and P. macrosporum Vaartaja & van der Plaats-Niterink. Cavity spot can be controlled with metalaxyl, but reports are emerging that this treatment show little or no efficacy in many regions. This situation reinforces the need for alternative fungicides. The objectives of the present study were: (1) to determine the sensitivity of 14 pathogenic isolates of P. sulcatum, P. sylvaticum and P. macrosporum collected from carrots produced in the area of the city of Québec to different broad-spectrum and oomycete-specific fungicides (chlorothalonil, etridiazole, fludioxonil, fosetyl-Al, metalaxyl, zoxamide), (2) to evaluate the efficacy of the fungicides in controlling cavity spot, and (3) to evaluate the risk of resistance development of isolates with the best-performing fungicide(s). The determination of EC50 for the fungicides tested showed that most isolates were highly sensitive to both metalaxyl and zoxamide but insensitive to fludioxonil, fosetyl-Al and chlorothalonil. In greenhouse assays, only zoxamide provided significant and consistent disease control as measured by the number of cavity spot lesions caused by P. sulcatum. Investigations into the risk of resistance development to zoxamide showed that, for specific isolates, repeated exposure to the fungicide resulted in a loss of sensitivity.     

Lateral roots of carrot have a low impact on alloinfections in cavity spot epidemic caused by <Emphasis Type="Italic">Pythium violae</Emphasis>

Carrot cavity spot, caused by a complex of Pythium species, is characterized by sunken elliptical lesions on the taproot. Recent epidemiological studies of P. violae have demonstrated the occurrence of both primary and secondary infections, with two types of secondary infection, autoinfection and alloinfection. Investigating the mechanisms underlying alloinfection and the role of carrot lateral roots, we asked whether direct physical root contact plays a role in alloinfection and whether root exudates enhance mycelial growth in soil alone. A rhizobox system was designed to differentiate the effects of each mechanism: a buffer zone created by nylon mesh was used to test the first mechanism, and young carrots with a root system similar to lateral roots were used to test the second. Alloinfections were generated in rhizoboxes via diseased taproots transplanted close to healthy, mature carrots. The nylon mesh had no significant effect on disease intensity (reflecting alloinfection), providing evidence that mycelial growth in soil contributed more to disease spread than did physical contact among roots. Nor did young carrots significantly affect alloinfection; thus root exudates had little effect on mycelial growth.     

PCR‐based identification of Pythium spp. causing cavity spot in carrots and sensitive detection in soil samples

On the basis of ITS sequences PCR primers were designed for the identification of the five Pythium species found to be most important for the development of carrot cavity spot in Norway: P. intermedium, P. sulcatum, P. sylvaticum, P. violae and P. ‘vipa’. The P. ‘vipa’ isolates had a unique ITS sequence, differed morphologically from all other Pythium isolates, and thus probably represent a new species. The PCR primers were species‐specific with no cross‐reaction to other Pythium species or to fungal isolates from carrot tested. The detection limits varied for the different primer pairs. The two most sensitive assays allowed detection of as little as 5 fg DNA. All five Pythium species could be detected in lesions from diseased carrots. Weak positive signals were obtained from some carrot samples without symptoms. PCR assays allowed detection of pathogens in soil. In samples of soil known to produce cavity spots on cropped carrots, strong signals were obtained. In several soil samples more than one of the five Pythium species could be detected. The utilization of this diagnostic PCR assay in analysis of field soil and carrot tissue might in the future be exploited to reduce the incidence of this serious carrot disease.     

Evaluation of carrot resistance to alternaria leaf blight in controlled environments

The objective of this study was to find a technique for plant resistance screening to alternaria leaf blight (ALB), caused by the fungus Alternaria dauci , in controlled environments. Glasshouse and laboratory screening methods were compared using three cultivars and F₂ genotypes segregating for ALB resistance evaluated against self-pollinated F₃ field-grown plants. Plant disease was assessed through a disease index obtained from the size and number of symptoms on carrot leaves. The results indicated the value of glasshouse evaluation and the inadequacy of detached leaf and hypocotyl assays for carrot screening for ALB resistance. Spearman's rank correlation, applied to results obtained with both F₂ plants and their progeny, indicated that the optimal evaluation stage for ALB resistance in carrot is 20 days after inoculation. This test was powerful enough to be used as a prescreening test in breeding programmes.     

Selection for resistance to the herbicide imazethapyr in somaclones of soyabean

Cultivars of soyabean [ Glycine max (L.) Merr.] resistant to the herbicide imazethapyr were identified by suspending the roots of 5-day-old seedlings in nutrient culture containing 2.5 mg a.i. L^–1 imazethapyr and then comparing the inhibitory effect on root length and shoot dry weight. The four most resistant cultivars were subsequently screened as regenerating tissue cultures in a medium containing 2.0 mg a.i. L^–l imazethapyr to select somaclonal cells with increased resistance. Surviving portions of cultures were regenerated to give shoots, the plants isolated, allowed to flower and seed. These progeny were then used for further seed multiplication and seedlings from this latter generation were exposed to imazethapyr in vivo and callus and cell suspension cultures derived from these seedlings were exposed to imazethapyr in vitro . A reduction in the inhibitory effect of the imazethapyr was noted in the somaclone seedlings and tissue cultures. However, measurement of acetolactate synthase (ALS) activity showed no differences among the parent cultivars and in the selected somaclones in this trait.     

Evaluation of different methods for the characterization of carrot resistance to the alternaria leaf blight pathogen (Alternaria dauci) revealed two qualitatively different resistances

Alternaria leaf blight (ALB), caused by Alternaria dauci, is one of the most damaging foliar diseases of carrot worldwide. The aim of this study was to compare different methods for evaluating levels of carrot resistance to ALB. Three techniques were investigated by comparison with a visual disease assessment control: in vivo conidial germination, a bioassay based on a drop‐inoculation method, and in planta quantification of fungal biomass by quantitative PCR (Q‐PCR). Three carrot cultivars showing different degrees of resistance to A. dauci were used, i.e. a susceptible cultivar (Presto) and two partially resistant genotypes (Texto and Bolero), challenged with an aggressive or a very aggressive isolate of A. dauci. Both partially resistant genotypes produced a higher mean number of germ tubes per conidium (up to 3·42±0·35) than the susceptible one (1·26±0·18). The drop‐inoculation results allowed one of the partially resistant genotypes (Bolero, log₁₀(S+1) = 1·34±0·13) to be distinguished from the susceptible one (1·90±0·13). By contrast, fungal growth measured by Q‐PCR clearly differentiated the two partially resistant genotypes with log₁₀(I) values of 2·77±0·13 compared to the susceptible cultivar (3·65±0·13) at 15 days post‐inoculation. This result was strongly correlated (r² = 0·91) with the disease severity index scored at the same date. Data obtained with the different assessment methods strongly suggest that the Texto and Bolero genotypes have different genetic resistance sources.     

Assessment of the inheritance of resistance and tolerance in cherry (Prunus sp.) to Blumeriella jaapii,the causal agent of cherry leaf spot

Cherry leaf spot (CLS), caused by Blumeriella jaapii, is a serious fungal disease of sour cherry (Prunus cerasus). Cultivar Montmorency, the major cultivar grown in the United States, is highly susceptible to CLS. As many as 10 fungicide sprays can be required each growing season to combat this disease; therefore, developing CLS‐resistant cultivars is a top breeding priority. Germplasm previously reported to be resistant or tolerant to CLS was acquired and incorporated into the sour cherry breeding programme at Michigan State University (MSU) and included three cherry species: sour cherry, sweet cherry (P. avium), and the wild species P. canescens. This study aimed to: (i) compare the CLS disease progression profile of the susceptible cultivar Montmorency with those of the resistant and tolerant germplasm; and (ii) gain an understanding of the inheritance of these resistance and tolerance traits by evaluating the host response of progeny individuals belonging to families derived from this germplasm. Significant differences were observed between the susceptible Montmorency and the tolerant and resistant accessions in their response to CLS and its progression during the growing season. Evaluation of the CLS host responses of progeny individuals derived from this germplasm supported a dominant two‐gene model for P. canescens‐derived resistance and a recessive gene model for sweet cherry‐derived tolerance. These insights into disease progression and trait inheritance improve the efficiency and potential success of breeding sour cherry cultivars with durable resistance to CLS.     

黄淮海地区夏玉米灰斑病病原菌鉴定及主栽品种抗性分析

 为明确黄淮海夏玉米区玉米灰斑病的病原菌种类,于2017年从河北、河南、山东、安徽、天津五省市疑似发生玉米灰斑病的76个市县采集样本,采用组织分离法和显微单孢分离法获得病原菌,通过形态与分子生物学手段进行病原菌种类鉴定,并对黄淮海地区、陕西、辽宁、黑龙江的37个菌株进行ISSR聚类分析。结果表明,引起黄淮海地区玉米灰斑病的病原菌为玉蜀黍尾孢(Cercospora zeae-maydis),玉蜀黍尾孢的变异具有地理种群特征。同时对黄淮海夏玉米区主栽品种进行人工接种鉴定,结果显示6个主栽品种中郑单958、登海605、先玉335、浚单20等4个品种对玉米灰斑病抗性较差,玉米灰斑病在本区域具有大面积发生和流行的风险。本研究为黄淮海夏玉米区灰斑病的综合防控提供支撑和理论依据。     

黄淮海地区夏玉米灰斑病病原菌鉴定及主栽品种抗性分析

 为明确黄淮海夏玉米区玉米灰斑病的病原菌种类,于2017年从河北、河南、山东、安徽、天津五省市疑似发生玉米灰斑病的76个市县采集样本,采用组织分离法和显微单孢分离法获得病原菌,通过形态与分子生物学手段进行病原菌种类鉴定,并对黄淮海地区、陕西、辽宁、黑龙江的37个菌株进行ISSR聚类分析。结果表明,引起黄淮海地区玉米灰斑病的病原菌为玉蜀黍尾孢(Cercospora zeae-maydis),玉蜀黍尾孢的变异具有地理种群特征。同时对黄淮海夏玉米区主栽品种进行人工接种鉴定,结果显示6个主栽品种中郑单958、登海605、先玉335、浚单20等4个品种对玉米灰斑病抗性较差,玉米灰斑病在本区域具有大面积发生和流行的风险。本研究为黄淮海夏玉米区灰斑病的综合防控提供支撑和理论依据。     

水稻新病害叶鞘黑斑病的病原鉴定

 2011年7月,在湖南省花垣县水稻上发现一种新的病害,症状表现为叶鞘上出现长椭圆形黑斑,病健交界模糊。分离到的4隔孢弯孢霉(HNHY001)经离体和活体人工接种均可产生黑斑;另一种黑孢霉菌株(HNHY002)不引致叶鞘黑斑。从湖南省栽培水稻150份种子样本中共分离到弯孢霉菌株27个,其中4个菌株具有4隔孢子。这4个菌株中1个菌株不产生子座,其余3个均产生分枝子座。经人工接种除不产生子座的外,其余3个菌株均能产生典型的叶鞘黑斑。HNHY001的rDNA-ITS序列(GenBank登录号JQ360963)经BLAST搜索,与之最接近的几个序列为膝曲旋孢腔菌(Cochliobolus geniculatus)及其有丝分裂产孢种—4隔孢弯孢霉。鉴于单独以及与其它4隔孢弯孢霉菌株进行对峙培养均未产生有性阶段,建议根据此菌无性阶段特征即分生孢子较直、4横隔、子座分枝等特征,鉴定为膝曲旋孢腔菌有丝分裂产孢种之一的假弯孢(C.fallax)。水稻叶鞘黑斑病系国内外首次报道。     

Effects of acid extracts of tomato (Lycopersicon esculentum Mill.) and carrot (Daucus carota L.)wastes from the food industry on the growth of some crops and weeds

The effects of acid extracts of tomato ( Lycopersicon esculentum Mill.) and carrot ( Daucus carota L.) juice wastes from the food industry on the growth of five species of crops and four weeds were examined. The acid extract of the tomato juice waste promoted the shoot and root growth of tomato, Chinese cabbage, corn, and radish, but not the growth of oat seedlings. The extract did not promote the shoot growth of weeds tested, and it inhibited the root growth of the weeds that included barnyardgrass ( Echinochloa oryzicola Ohwi), southern crabgrass ( Digitaria ciliaris Koel.) and Japanese barnyard millet ( Echinochloa utilis Ohwi). The carrot juice waste was also examined as another waste from the food industry. The effect of the acid extract of carrot juice waste showed significant promoting effects on the root growth of Chinese cabbage; however, the effects on other plants were lower than that of tomato juice waste. It also did not inhibit the growth of weeds. These results suggested that the acid extract from tomato juice waste is useful as plant-growth substances because they have a promoting effect on the shoot and root growth of crops, and an inhibitory effect on the root growth of some weeds.     

Pseudocercospora griseola,the causal agent of common bean angular leaf spot: Strain characterization and sensitivity to fungicides

Angular leaf spot (ALS), an important disease of common bean (Phaseolus vulgaris), is caused by the fungus Pseudocercospora griseola. This pathogen has a wide genetic variability and, therefore, poses a challenge to integrated disease management. The use of resistant cultivars is difficult; hence, the application of fungicides has been a common practice in common bean cultivation. P. griseola strains were morphophysiologically characterized and their sensitivity to common fungicides used to control ALS was studied. The strains were evaluated for sporulation capacity and a representative sample of 34 strains was bioassayed to determine their sensitivity to commercial concentrations of five fungicides, namely pyraclostrobin, mancozeb, pyraclostrobin + metconazole, chlorothalonil and tebuconazole. Another sample of 29 strains was studied for conidial germination and dimensions. Sporulation capacity ranged from 0.88 to 27.67 × 10⁴ conidia/ml and germination percentage ranged from 39% to 72%. The large differences among strains suggest a wide genetic variability among the strains. A wide variability in aggressiveness of P. griseola was observed, which has consequences for breeding programmes aimed at resistance. The behaviour of pathogen strains differed in every fungicide evaluated, even in a population that has not been under selection pressure in the field. These results confirm the need for further studies and may guide future research with this pathogen.