Production and evaluation of monoclonal antibodies for the detection of beet mild yellowing luteovirus and related strains

A sugar-beet-infecting isolate of beet mild yellowing luteovirus (BMYV), and aBrassica-infecting isolate of beet western yellows luteovirus (BWYV) were used to produce monoclonal antibodies for epidemiological studies with BMYV and related field strains. Thirty-four monoclonal antibodies were tested for their reaction with 9 luteoviruses in triple-antibody-sandwich enzyme-linked immunosorbent assay. One (MAFF 24) is now routinely used in the UK for detecting BMYV and BWYV in plants and aphids, although it does not discriminate between them. Heterologous reactions were detected between some of the monoclonal antibodies and potato leafroll virus (PLRV), bean leafroll virus (BLRV) and barley yellow dwarf virus (BYDV-RPV). 38% of antibodies raised to BWYV reacted with PLRV compared with 4% of those raised to BMYV. Monoclonal antibodies were produced which distinguished a sugar-beet-infecting isolate of BMYV with differing host range and serological properties from the commonly-occurring field strain.     

Histopathology of compatibility and incompatibility between oilseed rape and Albugo Candida

Cotyledons of one resistant and three susceptible rape lines/cultivars were inoculated with zoospores of Albugo Candida race 7. Samples of whole cotyledons were examined by differential interference contrast microscopy. The time course of the infection process was followed histologically. Germination of zoospore cysts occurred 2-3 h after inoculation. Infection was initiated with germ-tubes penetrating through stomata. Haustorium formation was first observed in the palisade mesophyll cells adjacent to the substomatal chambers 8 h after inoculation.
Only after the establishment of the first haustorium did compatible and incompatible interactions begin to differentiate. In the resistant cultivar, most primary hyphae produced single haustoria. Necrosis of the invaded host cell was first observed 12 h after inoculation followed by cessation of fungal growth. The death of host cells was largely restricted to the penetration site; the adjacent non-penetrated cells remained apparently unaffected. In the susceptible hosts, necrosis of infected cells occurred only infrequently, and hyphal growth continued unabated, resulting in mycelial ramification into the mesophyll. Numerous haustoria were produced.
Histological studies showed that the earliest event distinguishing a compatible from an incompatible interaction occurred after formation of the first haustorium and that resistance was not manifested until the host mesophyll cell had come into contact with the first haustorium. The distinction between compatibility and incompatibility was substantiated by quantitative analysis of white rust development on both resistant and susceptible lines/cultivars.     

The isolation and characterization of polyclonal and monoclonal antibodies to anastomosis group 8 of Rhizoctonia solani

Polyclonal and monoclonal antibodies were raised against secreted proteins from an anastomosis group 8 isolate of Rhizoctonia solani and tested for reactivity to field isolates from anastomosis groups 2-1, 3,4 and 8. Polyclonal antibodies raised against total secreted proteins cross-reacted in immunoblotting experiments with all R. solani isolates. However, immunoreactive proteins specific to Ag-8 isolates were evident. Monoclonal antibodies to secreted proteins were raised which reacted with fewer proteins and showed a greater degree of specificity for AG-8 isolates. Two monoclonals were selected for further study. One, an IgM monoclonal antibody, reacted with all R. solani isolates, recognizing a 40-kDa protein specific to AG-8 isolates and proteins of lower molecular weight in isolates from other anastomosis groups. The other, an IgG monoclonal antibody, was more specific, reacting with 38, 40 and 55-kDa proteins from AG-8 isolates and cross-reacting with few isolates from other anastomosis groups. Preliminary results on the induction of antigens recognized by the monoclonal antibodies are presented. The monoclonal antibodies characterized here are useful for the identification of isolates of R. solani and may also be used as probes to clarify the relationships between anastomosis groups.     

Microscopical studies of resistance to powdery mildew disease in the hop cultivar Wye Target

Microscopical studies of fungal development and host responses during infection of the hop cultivars Northern Brewer (susceptible) and Wye Target (resistant) with a Zenith isolate of Sphaerotheca humuli are described. Resistance to powdery mildew disease in cv. Wye Target is principally determined by the R₂ major gene. Fungal growth was typically restricted in cv. Wye Target following formation of a single haustorial initial or haustorium which failed to develop the characteristic lobes found in susceptible cells. The hypersensitive reaction of penetrated epidermal cells was associated with death of haustorial initials but the associated appressorium remained alive during the first 2 days after inoculation. In leaves expressing resistance, histochemical staining revealed deposition of lignin-like material and callose in penetrated cells and to a lesser extent in underlying palisade mesophyll cells. Transmission electron microscopy and enzymic digestion clearly demonstrated extensive paramural deposition of β-1.3 glucans (callose) in reacting mesophyll cells. Plant cell death, lignification and widespread callose deposition were rarely observed at infection sites in cv. Northern Brewer but collars of callose were deposited around the necks of all haustoria formed.     

Hypersensitive cell death and post-haustorial defence responses arrest the orange rust (Hemileia vastatrix) growth in resistant coffee leaves

The growth of a coffee orange rust fungus (Hemileia vastatrix Berk and Br.) isolate (race II) and the sequence of responses it induced in leaves of resistant Coffea arabica L. and C. congensis Froehner as well as on a susceptible C. arabica were investigated cytologically and biochemically. The percentages of germinated urediospores and of appressoria formed over stomata as well as the fungal growth inside leaf tissues were similar in resistant and susceptible leaves until the 3rd day after the inoculation. In the susceptible leaves, at the majority of the infection sites (70%) the fungus pursued its growth without apparent inhibition while in the resistant leaves the fungus ceased its growth with higher frequency (34% in C. arabica and 54% in C. congensis) after the formation of at least one haustorium. The first signs of incompatibility, detected 2 days after the inoculation, were cytologically expressed by hypersensitive host cell death (HR), host cell wall autofluorescence and haustoria encasement with callose and β-1,4-glucans. Biochemically, two peaks of phenylalanine ammonia-lyase (PAL) activity were detected by 2 and 5 days after the inoculation. The 1st peak coincided with the early accumulation of phenolic compounds and with the beginning of cell death. The 2nd peak could be related to later accumulation of phenols and the lignification of the host cell walls. About 5–7 days after the inoculation, ultrastructural observations revealed the accumulation of a material partially crystallized in the intercellular spaces around the senescent hyphae, next to dead host cells and in close association with the middle lamella that initially labelled for pectins. It also contained polysaccharides and phenolic-like compounds. Cellulose, hemicellulose, extensins, hydroxyproline-rich glycoproteins and proteins were not detected. The hypertrophy of the host cells in the infection area were also observed around 12 days after the inoculation corresponding macroscopically to the reaction flt.In susceptible plants, cell death was also observed 3 days after the inoculation but only in a reduced percentage of infection sites in which the fungus aborted at an early stage. A late haustorium encasement and stimulation of PAL activity were also observed but these delayed host responses did not prevent fungal growth and sporulation.The intercellular material, only observed in the resistant plants, is here reported for the first time and although its role is unknown it might be the result of plant cell death.     

Cytological events in tomato leaves inoculated with conidia of <Emphasis Type="Italic">Blumeria graminis</Emphasis> f. sp. <Emphasis Type="Italic">hordei</Emphasis> and <Emphasis Type="Italic">Oidium neolycopersici</Emphasis> KTP-01

Leaves of tomato and barley were inoculated with conidia of Blumeria graminis f. sp. hordei race 1 (R1) or Oidium neolycopersici (KTP-01) to observe cytological responses in search of resistance to powdery mildew. Both conidia formed appressoria at similar rates on tomato or barley leaves, indicating that no resistance was expressed during the prepenetration stage of these fungi. On R1-inoculated tomato leaves, appressoria penetrated the papillae, but subsequent haustorium formation was inhibited by hypersensitive necrosis in the invaded epidermal cells. On the other hand, KTP-01 (pathogenic to tomato leaves) successfully developed functional haustoria in epidermal cells to elongate secondary hyphae, although the hyphal elongation from some conidia was later suppressed by delayed hypersensitive necrosis in some haustorium-harboring epidermal cells. Thus, the present study indicated that the resistance of tomato to powdery mildew fungi was associated with a hypersensitive response in invaded epidermal cells but not the prevention of fungal penetration through host papilla.     

Fungal suppression of resistance against inappropriate Blumeria graminis formae speciales in barley,oat and wheat

When barley, wheat or oat leaf epidermal cells were attacked by their appropriate forma specialis (f.sp.) of Blumeria graminis DC. Speer (f.sp. hordei, tritici and avenae, respectively), many attempted penetrations succeeded, functional haustoria were formed and very few plant cells died. When attacked by either of the two possible inappropriate ff.spp., penetration attempts failed in association with papilla deposition by epidermal cells, attacked cells died, or if visible haustoria were formed the plant cell died very soon afterwards. Double inoculation experiments were performed where each cereal species was first attacked by its appropriate f.sp., as inducer, and later by the different ff.spp. as challenger. Infection by the appropriate inducer profoundly affected cellular responses to challenger attack. Suppression of defensive responses was dramatic within epidermal cells containing the inducer haustorium, evident to some extent in adjacent cells, but undetectable at two cells distance. Suppression of penetration resistance allowed most challenger attacks, even by inappropriate ff.spp., to form a haustorium. Furthermore, death of penetrated epidermal cells was also suppressed so that haustoria of the inappropriate ff.spp. functioned to support colony development. In oat, delayed epidermal cell death prevented full colony development by inappropriate ff.spp., but in barley and wheat, no cell death was apparent by four days after inoculation and colonies of the inappropriate ff.spp. produced extensive hyphae, secondary haustoria and conidial chains.     

Uromyces Viciae-fabae Haustorium Formation in Susceptible and Resistant Faba Bean Lines

Haustorium formation by the faba bean rust (Uromyces viciae-fabae) was studied on susceptible and resistant faba bean lines. The resistant lines showed incomplete resistance, based on late acting hypersensitivity or on non-hypersensitive resistance acting before haustorium formation. Histological observations on infected leaves showed that both the number of haustoria per infection unit and their developmental stage was reduced in both resistant lines. Isolation of haustoria confirmed that both the number and the size of haustoria were reduced in resistant lines, irrespective of whether the resistance was associated with hypersensitivity. Plant age had no detectable effect on both parameters.     

大豆疫霉菌对大豆下胚轴侵染过程的细胞学研究

 接种后1.5~24h,用光镜和电镜研究了2个大豆品种与大豆疫霉菌Ps411的亲和性和非亲和性互作。观察结果表明,大豆疫霉菌对大豆下胚轴的侵染过程可分为侵入前、侵入、皮层组织中的扩展和进入维管束组织4个连续阶段。大豆下胚轴接种后在25℃保湿培养,1.5h后游动孢子即形成休止孢并萌发产生附着孢,3h后侵入表皮细胞,6h后进入皮层组织,24h后进入维管束组织。病原菌主要以侵染菌丝直接侵入表皮,表皮细胞间隙是主要侵入部位。皮层细胞是病原菌定殖和发展的主要场所,胞间菌丝侵入皮层细胞并形成吸器。在菌丝与寄主细胞接触部位的寄主细胞壁与质膜之间常有胞壁沉积物的形成。在抗病品种上病菌的侵染事件与感病品种基本一致,但不能形成正常的吸器,胞壁沉积物明显多于感病品种,菌丝在寄主组织内的扩展明显受到抑制。利用β-1,3-葡聚糖免疫金标记单克隆抗体进行的免疫细胞化学的研究表明,胞壁沉积物内含有大量的β-1,3-葡聚糖,在大豆疫霉菌菌丝壁中也存在β-1,3-葡聚糖。以上结果表明,病原菌的侵染可诱导抗病寄主细胞内β-1,3-葡聚糖迅速的合成与积累、并形成胞壁沉积物,以抵御病菌的侵染与扩展。     

Development of monoclonal antibody-based immunological assays for the detection of live propagules of Rhizoctonia solani in soil

Mouse monoclonal antibodies (mAbs) and rabbit (polyclonal) antiserum were used to develop DIAGNOSTIC-ELISA, double-antibody-sandwich-ELISA (DAS-ELISA), DIP-STICK and immuno-fluorescence colony staining immunoassays for the specific detection of Rhizoctonia solani in soil. mAbs were raised against an anastomosis group 4 isolate of R. solani. Mice were immunized using either phosphate-buffered saline (PBS) suspensions of lyophilized mycelium plus Quil A adjuvant, or with a solubilized acetone precipitate prepared from cell-free surface washings from solid slant cultures. Polyclonal antisera were raised in rabbits using PBS suspensions of lyophilized mycelium and Quil A adjuvant. Hybridoma supernatants and rabbit antisera were screened by ELISA. Four of the cell lines raised produced mAbs that were species-specific. They recognized antigens from R. solani by ELISA and immunofluorescence, but not other related or unrelated species of soil-borne fungi. The remaining cell line produced mAbs that cross-reacted slightly, by ELISA, with antigens from R. cerealis. These mAbs did not recognize R. cerealis by immunofluorescence, or other related or unrelated soil-borne fungi, by ELISA.     

小麦与条锈病菌不亲和互作的超微结构

 采用条锈菌同一小种的野生型菌系和弱毒突变菌系,分别接种同一小麦品种的方法,研究了不亲和互作的超微结构特征。在不亲和互作中,条锈菌的胞间菌丝、吸器母细胞和吸器明显受抑。吸器可以在发育早期受抑坏死,也可迟滞至吸器体形成之后坏死。吸器外质膜严重皱褶,并出现孔洞,吸器外间质加宽,沉积大量电子致密物质。侵染位点的小麦叶肉细胞表现与过敏性坏死反应相关联的一系列变化。细胞壁内侧还出现乳突状或颗粒状沉积物等防御结构或物质。     

小麦新抗源一粒葡抗条锈病的组织学和超微结构研究

 采用荧光显微镜、微分干涉显微镜和电子显微镜技术,系统研究了小麦新抗源一粒葡抗小麦条锈病的组织学和超微结构特征。结果表明:相对于感病品种铭贤169,一粒葡对条锈菌的侵染,在组织学和超微结构上均表现出明显的抗性特征。在组织学水平,表现为菌丝生长受抑,菌落发育延迟或败育,吸器母细胞和吸器数目明显减少;同时,侵染点的寄主细胞表现出不同程度的过敏性坏死症状。电镜观察发现,在一粒葡和感病品种中,条锈菌均可由芽管顶端直接进入或通过形成附着胞进入小麦气孔。其后,在一粒葡上,病菌胞间菌丝、吸器母细胞、吸器在细胞和亚细胞水平均发生了一系列异常变化,表现为原生质染色逐渐加深,液泡增多变大,逐渐消解原生质;胞间菌丝、吸器母细胞细胞壁不规则增厚;胞间菌丝线粒体肿胀,数目增多,逐渐解体;吸器母细胞细胞质逐渐空泡化后丧失其生理功能;吸器外质膜皱褶;吸器外间质加宽并有丝状或颗粒状物质形成,吸器体壁逐渐消解出现孔洞,吸器体最终畸形坏死。同时,寄主细胞产生一系列显著的结构防卫反应:形成胞壁沉积物、乳突、吸器鞘等结构,以及发生坏死,阻碍并抑制病菌的发育及扩展。     

Characterization of Resistance Mechanisms to Erysiphe pisi in Medicago truncatula

ABSTRACT In this work, we studied the resistance of 277 Medicago truncatula accessions against powdery mildew and further characterized the defense mechanisms of resistant plants. Ten resistant accessions were selected according to macroscopic assessment. Histological studies showed a range of defense mechanisms, acting alone or combined, that impeded fungal development at different stages. Some accessions allowed a reduced spore germination frequency compared with that of the susceptible control. In others, the fungus was arrested at penetration stage due to papilla formation. Epidermal cells of several accessions were penetrated by the fungus but then hypersensitive response (HR) leading to cell death hampered fungal development. In some cases, cell death was very fast and no haustorium could be observed in epidermal cells, whereas in others, haustoria and secondary hyphae indicated a slow HR. Finally, in some accessions in which no HR was observed, colony growth was restricted through posthaustorial defense mechanisms. Characterization of defense mechanisms will be useful for further cellular and molecular studies to unravel the bases of resistance in this species in particular and in legume-powdery mildew interaction in general.     

Identification of Common Epitopes on a Conserved Region of NSs Proteins Among Tospoviruses of Watermelon silver mottle virus Serogroup

ABSTRACT The NSs protein of Watermelon silver mottle virus (WSMoV) was expressed by a Zucchini yellow mosaic virus (ZYMV) vector in squash. The expressed NSs protein with a histidine tag and an additional NIa protease cleavage sequence was isolated by Ni(2+)-NTA resins as a free-form protein and further eluted after sodium dodecyl sulfate-polyacrylamide gel electrophoresis for production of rabbit antiserum and mouse monoclonal antibodies (MAbs). The rabbit antiserum strongly reacted with the NSs crude antigen of WSMoV and weakly reacted with that of a high-temperature-recovered gloxinia isolate (HT-1) of Capsicum chlorosis virus (CaCV), but not with that of Calla lily chlorotic spot virus (CCSV). In contrast, the MAbs reacted strongly with all crude NSs antigens of WSMoV, CaCV, and CCSV. Various deletions of the NSs open reading frame were constructed and expressed by ZYMV vector. Results indicate that all three MAbs target the 89- to 125-amino-acid (aa) region of WSMoV NSs protein. Two indispensable residues of cysteine and lysine were essential for MAbs recognition. Sequence comparison of the deduced MAbs-recognized region with the reported tospoviral NSs proteins revealed the presence of a consensus sequence VRKPGVKNTGCKFTMHNQIFNPN (denoted WNSscon), at the 98- to 120-aa position of NSs proteins, sharing 86 to 100% identities among those of WSMoV, CaCV, CCSV, and Peanut bud necrosis virus. A synthetic WNSscon peptide reacted with the MAbs and verified that the epitopes are present in the 98- to 120-aa region of WSMoV NSs protein. The WSMoV sero-group-specific NSs MAbs provide a means for reliable identification of tospoviruses in this large serogroup.     

Effect of cold‐induced changes in physical and chemical leaf properties on the resistance of winter triticale (×Triticosecale) to the fungal pathogen Microdochium nivale

This study showed that several mechanisms of the basal resistance of winter triticale to Microdochium nivale are cultivar‐dependent and can be induced specifically during plant hardening. Experiments and microscopic observations were conducted on triticale cvs Hewo (able to develop resistance after cold treatment) and Magnat (susceptible to infection despite hardening). In cv. Hewo, cold hardening altered the physical and chemical properties of the leaf surface and prevented both adhesion of M. nivale hyphae to the leaves and direct penetration of the epidermis. Cold‐induced submicron‐ and micron‐scale roughness on the leaf epidermis resulted in superhydrophobicity, restricting fungal adhesion and growth, while the lower permeability and altered chemical composition of the host cell wall protected against tissue digestion by the fungus. The fungal strategy to access the nutrient resources of resistant hosts is the penetration of leaf tissues through stomata, followed by biotrophic intercellular growth of individual hyphae and the formation of haustoria‐like structures within mesophyll cells. In contrast, a destructive necrotrophic fungal lifestyle occurs in susceptible seedlings, despite cold hardening of the plants, with the host epidermis, mesophyll and vascular tissues being digested and becoming disorganized as a result of the low chemical and mechanical stability of the cell wall matrix. This work indicates that specific genetically encoded physical and mechanical properties of the cell wall and leaf tissues that depend on cold hardening are factors that can determine plant resistance against fungal diseases.     

小麦叶锈菌在感病寄主上发育的组织病理学和超微结构研究

 应用荧光显微技术、微分干涉技术和生物电镜技术,系统地研究了小麦叶锈菌在感病寄主上的发育过程及其超微结构特征。小麦叶锈菌在感病品种上的发育过程可分为几个明显的阶段,即孢子的萌发、附着胞的形成、气孔下囊的分化、初生菌丝和次生菌丝的形成和生长、吸器母细胞和吸器的形成、夏孢子床和夏孢子堆的产生以及夏孢子的形成。小麦叶锈菌的胞间菌丝呈丝状,生长和分枝通常沿寄主细胞壁进行。胞间菌丝与寄主细胞的接触诱导了吸器母细胞的分化,吸器母细胞在与寄主细胞壁的接触部位发育形成入侵栓,穿透寄主细胞壁后于细胞内形成吸器。胞间菌丝和吸器母细胞均含有双核,而成熟吸器则含有单核。经常规染色后,胞间菌丝和吸器母细胞的壁与隔膜均可分辨出由多层构成。     

Production of monoclonal antibodies to potato virus YNTN strain and their use for strain differentiation

Four mouse monoclonal antibodies (MAbs) against potato virus Y (PVY) were produced. MAb 4C1 reacted with four isolates of PVY^NTN and only very weakly with one isolate of the necrotic strain of PVY (PVY^N). It did not react with other isolates of the ordinary strain of PVY tested. MAb 2C9 reacted with all isolates tested and can be used to produce a specific diagnostic kit for routine PVY detection. Other MAbs had different specificities and reacted with isolates of various strains of PVY. MAbs did not react with seven other members of the Potyvirus group including potato virus A. A MAb-based ELISA, using MAb 4C1, was devised and shown to detect PVY^NTN specifically.     

青杨叶锈病菌(Melampsora larici-populina Kleb.)侵染过程的超微结构研究

 采用电子显微镜技术对青杨叶锈病菌(Melampsora larici-populina Kleb.)的侵染过程进行了研究。发现该菌夏孢子萌发产生1~3个芽管,且具较多的树杈状分枝。芽管由气孔侵入,侵入前不形成明显的附着胞或仅个别芽管形成附着胞。芽管侵入气孔后在气孔腔内形成气孔下囊,再分化出圆形的膨大体而产生1~2支初生菌丝。初生菌丝在寄主细胞间扩展,与叶肉细胞壁接触后分化出吸器母细胞,吸器母细胞中的细胞器与胞间菌丝相同,双核。吸器母细胞产生侵入钉侵入叶肉细胞内部形成吸器,成熟吸器由细长具颈环的管状颈部和膨大的吸器体组成,此时胞间菌丝在吸器母细胞处分化出次生菌丝,在叶肉细胞间扩展形成次生菌落,产生孢子堆。病菌在寄主细胞间隙或沿寄主细胞壁延伸时,寄主细胞仍保持正常状态。     

Normal germling development by Erysiphe graminis on cereal leaves freed of epicuticular wax

Experiments were conducted to test the hypothesis that recognition of the physical structure of epicuticular leaf waxes by Erysiphe graminis may be important to the development of normal germlings and the formation of functional appressoria. Comparisons of germination rates and characteristics of germling development by E. graminis f.sp. avenae , and in one experiment by f.sp. hordei , were made between intact cereal leaves and leaves from which the epicuticular waxes had been stripped away.
Overall, fungal development was very similar on intact and wax-free leaves: although germination rates were slightly, but significantly, lower, and lengths of appressorial germ tubes slightly greater, on stripped than intact leaves, a very similar proportion of germlings formed apparently normal appressoria in both cases. This was true for f.sp. avenae on first- and fifth-formed leaves of susceptible and adult plant resistant oats, and on barley and wheat first leaves, and for f.sp. hordei on first leaves of barley, oat and wheat. The appressoria formed on stripped leaves not only appeared normal, but also formed haustoria with at least the same frequency as on intact leaves; in several experiments, a higher proportion formed haustoria in stripped than intact leaves. Wax removal did not affect the adult plant resistance of oat cv. Maldwyn, which limits haustorium formation by appressoria, indicating that epicuticular wax was not involved in this resistance. It is concluded that the physical structure of epicuticular wax is not involved in the recognition processes leading to normal germling development.