Association of aster yellows subgroup 16SrI-B phytoplasmas with a disease of Rehmannia glutinosa var. purpurea

A new phytoplasma disease of Rehmannia glutinosa var. purpurea was observed in the Czech Republic in 1998. Infected plants showing severely proliferating shoots, leaves reduced in size with vein clearing and chlorosis, shortened internodes and virescent petals died in advanced stages of the disease. Electron microscopy examination of the ultra-thin sections revealed the presence of numerous polymorphic bodies in phloem tissue of leaf midribs and petioles. The disease was successfully transmitted from infected plant via a dodder bridge into periwinkle ( Catharanthus roseus ). The phytoplasma aetiology of this disease was further confirmed by polymerase chain reaction (PCR) using universal primers R16F2/R16R2. Restriction fragment length polymorphism (RFLP) analysis of amplification products indicated the presence of aster yellows related phytoplasmas (16SrI-B) in naturally infected samples of R. glutinosa var . purpurea and in symptomatic periwinkle after dodder transmission of the agent. A comparison of the amplified sequence with 17 sequences available in the GenBank confirmed the classification of the phytoplasma in the subgroup 16SrI-B. This is the first report of natural occurrence of phytoplasma-associated disease in R. glutinosa var. purpurea.     

Electron microscopy and molecular identification of phytoplasmas associated with strawberry green petals in the Czech Republic

The presence of phytoplasma inFragaria ananassa x Duch cv Senga Sengana showing strawberry green petals symptoms was observed by electron microscopy of phloem tissue. No phytoplasmas were found in asymptomatic strawberry plants used as controls. Nucleic acids extracted from these plants were used in nested-PCR assays with primers amplifying 16S rRNA sequences specifie for phytoplasmas. Bands of 1.2 kb were obtained and the subsequent nested-PCR with specific primers and RFLP analyses allowed to classify the detected phytoplasmas in the aster yellows group (16SrI). They belonged to the subgroup I-C of which type strain is clover phyllody phytoplasma.     

丁香卷叶病植原体的分子鉴定

 通过透射电子显微镜,在表现卷叶、褪绿症状的丁香(Syringa oblata)样品的叶脉韧皮部筛管细胞内观察到大量植原体粒子。应用植原体16S rRNA基因通用引物对P1/P7和R16F2n/R16R2对表症丁香植株总DNA进行巢式PCR扩增,得到了约1.2 kb的目标片段,通过对扩增片段进行测序、系统发育分析和同源性分析,结果表明,该片段长度为1 246 bp,在系统发育进化树上与翠菊黄化组（Candidatus Phytoplasma asteris）成员是聚集在一起的,与该组成员同源性均在98%以上。用16Sr RNAⅠ组和Ⅴ组特异引物确定了该病害非混合侵染所致,相似性系数和RFLP分析表明该植原体属于16SrⅠ B亚组。这是国内关于翠菊黄化组植原体在丁香上感染的首次报道。     

Role of wild Prunus species in the epidemiology of European stone fruit yellows

Several uncultivated trees of the species Prunus spinosa , P. cerasifera and P. domestica , sampled both adjacent to European stone fruit yellows (ESFY)-infected orchards and in isolation from cultivated stone fruit plants, were found to be infected by ESFY phytoplasma. These species were also colonized by Cacopsylla pruni , vector of the ESFY agent. In contrast, uncultivated species of Prunus avium , P. cerasus and P. mahaleb hosted neither the pathogen nor the vector. Insect- and graft-transmission trials of ESFY phytoplasma conducted under controlled conditions confirmed the data obtained in the field. The role played by the wild Prunus species is discussed and appears to be fundamental in the epidemic cycle of the disease.     

Molecular characterization ofPseudomonas syringae pv.tomato isolates from Tanzania

Bacterial speck caused byPseudomonas syringae pv.tomato is an emerging disease of tomato in Tanzania. Following reports of outbreaks of the disease in many locations in Tanzania, 56 isolates ofP. syringae pv.tomato were collected from four tomato- producing areas and characterized using pathogenicity assays on tomato, carbon source utilization by the Biolog Microplate system, polymerase chain reaction and restriction fragment length polymorphism (RFLP) analysis. All theP. syringae pv.tomato isolates produced bacterial speck symptoms on susceptible tomato (cv. ‘Tanya’) seedlings. Metabolic fingerprinting profiles revealed diversity among the isolates, forming several clusters. Some geographic differentiation was observed in principal component analysis, with isolates from Arusha region being more diverse than those from Iringa and Morogoro regions. The Biolog system was efficient in the identification of the isolates to the species level, as 53 of the 56 (94.6%) isolates ofP. syringae pv.tomato were identified asPseudomonas syringae. However, only 23 isolates out of the 56 (41.1%) were identified asPseudomonas syringae pv.tomato. The results of this work indicate the existence ofP. syringae pv.tomato isolates in Tanzania that differ significantly from those used to create the Biolog database. RFLP analysis showed that the isolates were highly conserved in theirhrpZ gene. The low level of genomic diversity within the pathogen in Tanzania shows that there is a possibility to use resistant tomato varieties as part of an effective integrated bacterial speck management plan. http://www.phytoparasitica.org posting August 8, 2008.     

Seasonal distribution of phytoplasmas in Australian grapevines

The distribution and persistence of phytoplasmas were determined in Australian grapevines. Phytoplasmas could be detected using the polymerase chain reaction (PCR) from shoots, cordons, trunks and roots throughout the year, and phytoplasmas appear to persistently infect Australian grapevines from year to year. Phytoplasmas were not always detected in samples from the same sampling area from one sampling period to the next. Phytoplasma detection by PCR was improved by sampling from shoots, cordons and trunks, especially during October (early spring). The diseases expressed by the 20 grapevines used in the distribution and persistence studies were monitored. Australian grapevine yellows disease (AGY) was expressed by 17/20 grapevines at some time during the study, whilst only 4/20 and 15/20 grapevines expressed restricted growth disease (RG) and late season leaf curl disease (LSLC), respectively. All grapevines with RG and LSLC also had AGY. The three diseases were persistently expressed in some grapevines and remission of disease was observed in others. The results of PCR detection in the same grapevines indicated that phytoplasmas were more frequently detected in AGY-affected grapevines that also expressed RG and LSLC compared with grapevines expressing AGY alone. Phytoplasmas were detected in symptomless plant material but less frequently compared with AGY-affected material.     

我国几种植物植原体的快速分子鉴别与鉴定的研究

 选用桑萎缩病(Mulberry dwarf,MD)、枣疯病(Jujube witches'-broom,JWB)、酸枣丛枝病(Wild jujube witches'-broom,WJWB)、泡桐丛枝病(Paulownia witches'-broom,PaWB)和苦楝丛枝病(Chinaberry tree witches'-broom,CWB)5种不同植物植原体和来源于3个不同地区PaWB和JWB材料进行16S rDNA和23S rDNA PCR扩增、异源双链迁移率分析(HMA)、PCR产物的RFLP分析和16S rDNA的克隆和测序等比较研究,建立了一种快速确定未知植原体种类和分类地位的分子鉴别与鉴定优化程序;并可对田间采集的各种植物植原体样品进行快速鉴定和鉴别。16S rDNA PCR产物HMA分析结果显示,JWB与CWB、MD和PaWB皆可形成明显的异源杂交双链;而CWB、MD和PaWB植原体之间未能形成异源双链。JWB和PaWB不同地区样品之间、JWB和WJWB之间也未发现异源杂交双链的形成。而23S rDNA PCR产物HMA分析则可以将MD与PaWB区分开。进一步对未知分类地位的CWB序列测定及与其它植原体16S rDNA的RFLP和同源性比较结果显示,CWB与PaWB同源性为99.5%,其中与MD的同源性高达99.7%,因而应将CWB归为翠菊黄花组16Sr I-B,16Sr I-B (rp-B)。     

Partial characterization of phytoplasmas associated with lettuce and wild lettuce phyllodies in Iran

Phytoplasmas associated with lettuce phyllody (LP) and wild lettuce phyllody (WLP) in southern Iran were partially characterized by molecular analyses and host-range studies. Agents of both diseases were transmitted by Neoaliturus fenestratus , a leafhopper colonizing lettuce and wild lettuce, to lettuce, wild lettuce, sowthistle and periwinkle, but not to safflower, sunflower, calendula and sesame. Both phytoplasmas induced bud proliferation, virescence, phyllody and witches' broom in infected plants. Total DNA extracted from infected lettuce and wild lettuce or from vector tissues was subjected to PCR using phytoplasma-specific primer pair P1/P7 or nested PCR using P1/P7 followed by R16F2n/R16R2. PCR product of nested PCR (1·2 kbp) was subjected to restriction fragment length polymorphism (RFLP). RFLP analysis of nested PCR product identified the LP, WLP and N. fenestratus -associated phytoplasmas as members of the pigeon pea witches' broom group, 16SrIX. Phylogenetic analysis of the 16S rRNA gene sequence also clustered LP and WLP phytoplasmas with other known members of the 16SrIX group. While no significant differences could be detected between LP and WLP phytoplasmas, both isolates differed from Lebanese wild lettuce phyllody in molecular properties.     

Molecular identification of Echinochloa oryzicola Vasing. and E. crus-galli (L.) Beauv. using a polymerase chain reaction–restriction fragment length polymorphism technique

Polymerase chain reaction (PCR) and polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) techniques were applied for establishing the reliable practice in identification of Echinochloa oryzicola Vasing. and E. crus-galli (L.) Beauv. (barnyardgrass). Total DNA was extracted from 18 accessions and 86 individuals of E. oryzicola , 33 accessions and 140 individuals of E. crus-galli var. crus-galli , 23 individuals of E. crus-galli var. praticola , and six individuals of E. crus-galli var. formosensis that were collected from Japan. A partial region of intergenetic spacer between trn T and trn L, and an intron of trn L were amplified separately using a trn-a and trn-b1 primer set, and a trn-c and trn-d primer set, respectively. All individuals of E. oryzicola showed the same fragment amplified by the trn-a and trn-b1 primer set. The fragment was 481 bp in length, and was undigested by Eco R I, whereas all individuals of E. crus-galli , including three botanical varieties, showed the same fragment with a 449-bp length. The fragment was digested by Eco R I into two fragments (178 and 271 bp). The fragment amplified by the trn-c and trn-d primer set in all individuals of E. oryzicola was digested by Alu I into two fragments (174 and 452 bp), but undigested by Dra I. In contrast, the fragment amplified by the trn-c and trn-d primer set in all individuals of E. crus-galli was digested by Dra I into two fragments (134 and 487 bp), but undigested by Alu I. There was no intraspecific variation in these regions; thus, these two species are easily identifiable by using our method.     

Hydrogen peroxide localization and antioxidant status in the recovery of apricot plants from European Stone Fruit Yellows

Hydrogen peroxide (H₂O₂) localization and roles of peroxidases, malondialdehyde and reduced glutathione were compared in leaves of apricot (Prunus armeniaca) plants asymptomatic, European Stone Fruits Yellows (ESFY)-symptomatic and recovered. Nested PCR analysis revealed that Candidatus Phytoplasma prunorum, is present in asymptomatic, symptomatic and recovered apricot trees, confirming previous observations on this species, in which recovery does not seem to be related to the disappearance of phytoplasma from the plant.H₂O₂was detected cytochemically by its reaction with cerium chloride, which produces electron-dense deposits of cerium perhydroxides. H₂O₂was present in the plasmalemma of the phloem cells of recovered apricot plant leaves, but not in the asymptomatic or symptomatic material. Furthermore, by labelling apricot leaf tissues with diaminobenzidine DAB, no differences were found in the localization of peroxidases.Protein content in asymptomatic, symptomatic and recovered leaves was not significantly different from one another. In contrast, guaiacol peroxidase activity had the following trend: symptomatic > recovered > asymptomatic, whereas reduced glutathione content followed the opposite trend: asymptomatic > recovered > symptomatic. Moreover, no differences were observed in malondialdehyde concentrations between asymptomatic, symptomatic and recovered leaves. The overall results suggest that H₂O₂ and related metabolites and enzymes appear to be involved in lessening both pathogen virulence and disease symptom expression in ESFY-infected apricot plants.     

Rapid diagnosis of sulfonylurea‐resistant Schoenoplectus juncoides [Roxb.] Palla using polymerase chain reaction–restriction fragment length polymorphism and isogene‐specific direct sequencing

Rapid diagnostic methods to detect known mutations in acetolactate synthase (ALS) genes that confer sulfonylurea (SU) resistance to Schoenoplectus juncoides were developed in this study. By using 11 SU‐resistant accessions (nine accessions with a Pro₁₉₇ substitution in ALS1 or ALS2, one accession with an Asp₃₇₆Glu substitution in ALS2 and one accession with a Trp₅₇₄Leu substitution in ALS2), polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analysis for DNA fragments that were amplified simultaneously from genomic ALS1 and ALS2 and PCR–RFLP analysis for DNA fragments that were amplified from either of the genomic ALS1 or ALS2 were carried out. In each of the two PCR–RFLP analyses, a common PCR product was digested separately with the restriction enzymes, BspLI, MboI and MunI, in order to detect Pro₁₉₇ substitutions, an Asp₃₇₆Glu substitution and a Trp₅₇₄Leu substitution, respectively. In each of the lanes where the detection of SU‐resistant substitutions was aimed, a specific band to suggest the existence of the said substitutions was observed in theoretically assumable ways. Separately, a direct sequencing method also was established, which was able to selectively sequence ALS1 or ALS2 from common templates containing both ALS1 and ALS2 by the isogene‐selective primers that were designed to anneal either of the ALS genes. It is expected that these methods could be used for the genetic analysis of SU‐resistant S. juncoides by providing rapid and accurate diagnosis.     

PCR法证实棉铃虫核型多角体病毒对棉铃虫经卵和蛹的垂直传播

根据棉铃虫核型多角体病毒的多角体蛋白基因设计5’引物和3’引物，通过PCR技术成功扩增389bp的目的片段，并对该片段进行了克隆和测序。结果表明，扩增片段为棉铃虫核型多角体病毒多角体蛋白基因序列。该检测方法的灵敏度为10fg，应用此技术从带毒的虫卵和蛹的总DNA中也检测到该病毒的存在。该方法从分子水平上证明了病毒的垂直传递。     

Comparative study of morphological,cultural and molecular markers for the characterization ofPseudocercosporella herpotrichoides isolates

Nirenberg's classification system and the polymerase chain reaction (PCR) combined with restriction enzyme digestion of an amplified ribosomal DNA fragment, were compared for the characterization of sixty isolates ofPseudocercosporella herpotrichoides, from various geographical areas and with differing fungicide sensitivity. With Nirenberg's system, it was possible to identify most isolates asP. herpotrichoides var.herpotrichoides orP. herpotrichoides var.acuformis. However, identification was slow and sometimes inconclusive as overlap occurred between the two varieties for all criteria examined. Molecular markers identified two distinct types among the isolates tested and generally good correlation was found between the PCR-based assay and Nirenberg's system, but the molecular assay was more accurate and faster.     

Epidemiological aspects of mango malformation disease caused by Fusarium mangiferae and source of infection in seedlings cultivated in orchards in Egypt

Mango malformation, caused by the fungus Fusarium mangiferae , is one of the major diseases of this crop occurring worldwide. This study was conducted to investigate aspects of the epidemiology, survival and spread of the pathogen in general and specifically in seedlings, the majority of which are cultivated in infected orchards in Egypt. Survival of conidia of a representative isolate (506/2) declined very rapidly in soil under summer conditions (1·6 weeks for 50% population decline), but significantly less in controlled and winter conditions (17·9 and 15·0 weeks, respectively, for 50% population decline). Likewise, inoculum survival in naturally infected panicles on the soil surface declined faster than in those buried at 30-cm depths. Natural infections were evaluated on fruits and seeds in a heavily infected and a healthy orchard. In infected trees, the skins of all sampled fruits within a 2-m radius of infected panicles were infected, but the pathogen was not detected in the seeds, seed coats or flesh. The pathogen was not detected in any parts of fruits from a healthy orchard. Vegetatively malformed mango seedlings, growing under infected trees bearing infected panicles, were sampled in two locations in Egypt to determine whether infection in seedlings was systemic (evenly distributed within plant tissue) or whether the pathogen originated from malformed panicles. According to PCR-specific primer amplification, the pathogen was detected in 97% of seedling apical meristems, declining gradually to 5% colonization in roots. It was concluded that inoculum of the pathogen originates from infected panicles and affects seedlings from the meristem, with infections descending to lower stem sections and roots. Minor infections of roots may occur from inoculum originating from infected panicles, but the pathogen is not seedborne.     

Identification of fungal pathogens and analysis of genetic diversity of Fusarium tricinctum causing root rots of alfalfa in north-east China

Alfalfa root rot is a devastating disease complex found worldwide. Population structure and genetic diversity of fungal pathogens causing alfalfa root rot in north-east China are not well understood. In this study, 480 fungal isolates were collected from six major alfalfa-growing regions in Heilongjiang province, China. They were identified as Fusarium tricinctum, F. oxysporum, F. acuminatum, F. solani, F. equiseti, Phoma medicaginis, Plectosphaerella cucumerina, Alternaria alternata, and Chaetomium globosum and caused root rot on alfalfa in greenhouse studies. F. tricinctum was the predominant species among the isolates, and P. medicaginis and C. globosum had not previously been reported causing alfalfa root rot in north-east China. Of the 73 F. tricinctum isolates identified, the majority were moderately or highly aggressive on alfalfa. No isolate of F. tricinctum was sensitive to carbendazim (1 and 10 μg/ml), indicating that, although commonly used, it is not suitable for management of the disease in this area. F. tricinctum isolates were analysed using AFLP markers and divided into eight genetic groups with 28 pairs of primers. Analysis of molecular variance indicated significant correlation between genetic groups of F. tricinctum isolates and their geographical locations or aggressiveness. Pairwise comparison and STRUCTURE analysis also indicated that geographical locations and aggressiveness of isolates had a significant effect on population differentiation. This study provides insight into the genetic diversity and reproductive biology of F. tricinctum, enhances understanding of the population diversity of alfalfa root rot pathogens in north-east China, and facilitates development of effective strategies for managing this destructive disease complex.     

谷子种子白发病菌带菌检测及种群多态性分析

为检测不同地区谷子种子携带白发病菌Sclerospora graminicola的情况以及种群多态性,以我国谷子不同主产区收集的95份代表性谷子品种种子为材料,提取种子基因组DNA并作模板,利用白发病菌28S rRNA序列设计特异性引物进行PCR扩增,将所获得的PCR产物测序后与白发病菌28S rRNA序列进行比对。结果表明,特异性引物Sg-28S-403-F/Sg-28S-1221-R对白发病菌具有高度的特异性,扩增出819 bp的特异性目标片段,有效检测灵敏度为0.125 ng/μL。在95份谷子种子中,26份种子检测出特异性扩增条带,所有扩增条带对应序列与白发病菌28S rRNA序列的相似性达98%以上,并且带菌种子全部来自春谷区。分析26份阳性样品的28S rRNA序列,得到58个变异位点和34种单倍型,其中单倍型SG2出现频率最高,内蒙古、陕西、山西、河北和辽宁省区的白发病菌都具有该单倍型,表明不同地理来源的白发病菌28S rRNA序列存在差异,SG2为优势单倍型。     

Characterization and PCR-based detection of benzimidazole-resistant isolates of Monilinia laxa in California

Monilinia laxa is a pathogen of brown rot of stone fruit and almond in California, causing blossom blights and fruit rots. In this study, low-level resistance to the benzimidazole fungicides benomyl and thiophanate-methyl was detected in field isolates of M laxa collected from stone fruits and almonds in California. Low-resistant (LR) isolates grew in potato dextrose agar (PDA) plates amended with benomyl and thiophanate-methyl at 1 and 5 microg ml(-1), respectively, but not in plates amended with benomyl at 5 microg ml(-1) or thiophanate-methyl at 50 microg ml(-1). The benzimidazole LR isolates were characterized by temperature sensitivity and the DNA sequence of the beta-tubulin gene. The LR isolates showed high-temperature sensitivity, being sensitive to 1 microg ml(-1) of benomyl at 28 degrees C but resistant at 8-24 degrees C. Analysis of the DNA sequence of the beta-tubulin gene showed that the LR isolates had a point mutation at the amino-acid position 240, causing substitution of leucine by phenylalanine. Based on the point mutation, a pair of allele-specific PCR primers was developed for rapid detection of LR isolates of M laxa. In addition, a pair of PCR primers specific to M laxa was developed on the basis of the differences in the DNA sequence of the intron 6 of beta-tubulin gene from M laxa, M fructicola and other fungal species. The primer pair amplified the expected 376-bp DNA fragment from all M laxa isolates tested, but not from 14 other fungal species isolated from stone fruit and almond crops. The restriction endonuclease BsmA I recognized the sequence GTCTCC in the PCR products from sensitive (S) isolates only, but not the GTTTCC sequence in the PCR products from LR isolates. The endonuclease digested the 376-bp PCR products from S isolates to produce two bands (111 and 265 bp) on agarose gels. Thus, both allele-specific PCR and the PCR-restriction fragment length polymorphism (PCR-RFLP) methods could be useful for rapidly detecting benzimidazole-resistant isolates of M laxa from stone fruit and almond crops in California.     

Detection of viral antigen in semi-thin sections of plant tissue by immunogold-silver staining and light microscopy

The immunogold-silver staining technique was developed for the light microscopical localization of viral antigen in plant tissue. Semi-thin sections of LR White-embedded plant tissue were immunologically labelled with primary antiserum and protein A-gold. Individual gold particles were covered with a silver precipitate using a physical developer. This precipitate could be seen as black spots in a conventional light microscope with brightfield and as brilliant white spots with darkfield illumination. Maximal sensitivity and low background was obtained when immunogold-labelled sections were fixed in glutaraldehyde prior to silver enhancement. Simultaneous observation of the silver coated gold label and cell morphology was achieved by epipolarization microscopy. Using this technique cowpea chlorotic mottle virus coat protein was detected in cowpea plants as function of the infection period. Virus translocation and multiplication was monitored in systemically inoculated tissue, showing viral antigen in phloem parenchyma of petiolules 6 h after systemic inoculation and subsequent spreading from the phloem to the neighbouring bundle sheath and cortex cells.Samenvatting De immunologische techniek (IGSS), waarbij complexen van antilichamen met proteïne A geadsorbeerd aan kolloïdaal goud (pAg) worden bedekt met zilver, werd met succes toegepast voor het aantonen van viraal antigeen in geïnfecteerd planteweefsel met behulp van de lichtmicroscoop. Semi-dunne plakjes weefsel werden ingebed in LR White en behandeld met antiserum tegen het cowpea chlorotic mottle virus (CCMV). Aan dit antigeen-antilichaam complex werd pAg gehecht. Vervolgens werd op de individuele gouddeeltjes zilver neersgeslagen met een ontwikkelaar bestaande uit een mensel van zilverlactaat en hydroquinone. De gouddeeltjes katalyseren de reductie van de zilverionen in oplossing tot metallisch zilver, dat neerslaat op de gouddeeltjes. Het zilverprecipitaat is waarneembaar als zwarting in een lichtmicroscoop met doorvallend licht en licht wit op bij donkerveld belichting. Maximale gevoeligheid van detectie en lage achtergrondkleuring werden bereikt door fixatie van het antigeen-antilichaam-pAg complex met glutaaraldehyde vóór de zilverkleuring.Gelijktijdige waarneming van het zilver label en de morfologie van de cellen was mogelijk door toepassing van gepolarisserd licht in een microscoop met opvallende belichting (epipolarisatiemicroscopie) in combinatie met doorvallend licht. Het zilverprecipitaat is hierbij waarneembaar als een helder blauwe kleur door de weerkaatsing en verstrooiing van het gefiltreerde gepolariseerde licht, terwijl de morfologie van de cytochemisch gekleurde cellen zichtbaar is met doorvallende belichting. Met IGSS in combinatie met epipolarisatiemicroscopie werd het CCMV gelokaliseerd in cowpea planten als functie van de infectieduur. De translocatie en vermenigvuldiging van het virus werden gevolgd in planteweefsel dat systemisch was geïnoculeerd volgens de differentiële-temperatuur-inoculatietechniek. Zes uur na systemische inoculatie werd het virus voor het eerst waargenomen in enkele floeemparenchymcellen en de infectie breidde zich daarna snel uit. Vierentwintig uur na inoculatie kon virus worden aangetoond in grote delen van het floeem, in de bundelschede en in de aangrenzende delen van de schors. Concluderend kan worden gesteld dat het virus vanuit de primaire bladeren naar de secundaire bladeren werd getransporteerd via het floeem, analoog aan het transport van assimilaten.Tot slot werden dunne plakjes geïnfecteerd weefsel, geïncubeerd met antiserum en proteïne A-goud, na zilverbehandeling vergeleken in licht-en elektronenmicroscoop.     

Pathology of cortical invasion by Plasmodiophora brassicae in clubroot resistant and susceptible Brassica oleracea hosts

The differences in physiological response of Brassica oleracea to infection and growth of Plasmodiophora brassicae in infected tissue were studied in clubroot resistant and susceptible hosts, grown in sand solution culture artificially inoculated with spores of P. brassicae (10⁸ spores mL⁻¹). Primary (root hair) and secondary (cortical) stages of P. brassicae occurred in both resistant and susceptible hosts. Symptoms of cortical invasion by P. brassicae in resistant and susceptible hosts included cell wall breaks, presence of vesicles or inclusion bodies within the cell walls, cell wall thickening in association with plasmodesmata and enlarged and/or disorganized host nuclei. The main difference between the resistant and susceptible host reaction was the absence of degradation of the secondary thickening and cell walls of the xylem in the resistant host. This study supports the existence of an amoeboid form of the pathogen in addition to the recognized two-phase life history of P. brassicae. Furthermore, it suggests that resistance in B. oleracea does not prevent the development of this amoeboid form. A reduced number of cell wall breakages suggests movement of the amoeboid form, may have been restricted but not prevented in the resistant host.