EXPERIMENTAL STUDIES OF HAUSTORIUM INITIATION AND EARLY DEVELOPMENT IN AGALINIS PURPUREA (L.) RAF. (SCROPHULARIACEAE)

In parasitic angiosperms the haustorium, an organ specialized for attachment and penetration of host tissue, functions in the transport of water and nutrients from the host to the parasite. In Agalinis purpurea (L.) Raf. (Scrophulariaceae) these organs are initiated laterally along its roots, opposite a primary xylem pole. Analyses of haustoria distribution and cellular root profiles show that the portion of the root which is most sensitive to haustorial elicitor molecules is the area distal to the zone of elongation and near the root meristem. Sectioned material supports this finding and, further, indicates that the cells which are the first to respond to haustorial elicitors are located in the inner cortex. Haustoria develop rapidly in response to a host root or to isolated chemical elicitors (xenognosins) normally contained in host root exudate. By 6 hr, vacuolation and radial cellular enlargement are observed in the cortex, and a lateral swelling along the root is visible. By 12 hr, cells of the epidermis divide anticlinally to establish a group of densely cytoplasmic cells at the apex of the haustorial swelling. Accompanying these divisions is the differentiation of specialized hair cells which elongate from epidermal cells flanking the presumptive haustorial apex. Next, the internal, radially enlarged cortical cells divide periclinally. Periclinal divisions are subsequently initiated in the pericycle as early as 18 hr post-induction. Cellular division and enlargement continue so that by 24–36 hr a mature pre-contact haustorium is formed. There is a reduction in root elongation concomitant with haustorial initiation. Depending upon the number of haustoria produced, elongation typically returns to the preinduction level within 2 or 3 days.     

桔梗胚乳吸器的细胞化学研究

对桔梗的胚乳吸器进行了细胞化学研究,结果显示,胚乳吸器的细胞质、胚乳吸器周围解体的珠心细胞和珠被细胞均呈强ＰＡＳ正反应。随着胚乳吸器的发育,吸器附近的珠心细胞和珠被细胞中贮存的大量淀粉粒逐渐减少和消失。胚乳吸器的细胞质,尤其是与胚乳本体细胞交界处的细胞质富含蛋白质。在球形胚前期,胚乳细胞中已积累大量的蛋白质颗粒。结果表明胚乳吸器起营养物质的吸收和转运作用,向胚乳提供养料。     

Studies on new types of haustoria in some Central EuropeanRhinanthoideae (Scrophulariaceae)

Two new types of haustoria have been found in many representatives ofRhinanthoideae. Metahaustoria are stimulated by host roots but lack contact with them. They develop a massive haustorial core with a large- and a smalled-celled portion, but little vascular core. In very old metahaustoria the vascular core is much enlarged and numerous tracheal elements develop in the reduced haustorial core and cortex. Warthaustoria are very small, occur only in the upper portions of the root system, and have host contacts. Vascular and haustorial core, and the endophyte are lacking, the intrusive cells are usually not endogenous, but rather roothair-like. Wart-haustoria may represent reduced and partly inhibited normal haustoria.     

Molecular Specificity of Haustorial Induction in Agalinis purpurea (L.) Raf. (Scrophulariaceae)

An exogenous signal normally contained in host root exudateis required for initiation of the haustorium by the root parasiteAgalinis purpurea (L.) Raf. (Scrophulariaceae). Two flavonoidsthat induce haustoria have been isolated from gum tragacanthand a number of structural analogues have been synthesized.The results show that a high degree of molecular specificityis required for haustorial induction. Both isolated flavonoidscontain substituted 3-methoxyphenol functionality, and syntheticanalogues have shown that 4-substituted 3-methoxyphenol functionalityis critical for high levels of haustorial induction. These dataprovide a model for understanding host recognition at the levelof haustorial induction in parasitic angiosperms. Agalinis purpurea (L.) Raf. Scrophulariaceae, haustorial induction, flavonoids, molecular specificity, parasitic angiosperms, xenognosin     

Analysis of In Vivo Protein Synthesis and Histological Studies of Haustorial Formation in Root Cultures of Witch weed (Striga asiatica L Kuntze)

We recently described an in vitro approach that uses root culturesto study haustorial formation in Striga asiatica. Previous studieshave used haustoria formed on intact radicles of Striga seedlings.In vitro cultured roots formed haustoria that appeared morphologicallysimilar to those formed by Striga radicles, but were 5–10-foldlarger. In this study, we provide biochemical and histologicalevidence to support further the similarity of root culture haustoriato haustoria formed on radicles of seedlings. We examined invivo protein synthesis during haustorial development on rootcultures and radicles by 2-D PAGE. Four proteins increased inabundance in both root cultures and radicles after 6 h of haustorialinduction. All four proteins appeared transiently in root culturesand radicles, being more abundant at 6 h, and less abundantafter 24 h of haustorial induction. Only three of the four haustorial-specificproteins were more abundant in root cultures after 2 h of haustorialinduction; all four had decreased in abundance after 12 h ofhaustorial induction. Using light microscopic analysis we comparedthe ontogeny of root culture haustoria to that of haustoriaon radicles. These studies revealed that root culture haustoriaundergo developmental changes similar to those reported forradicle haustoria such as early expansion of cortical cells,the emergence of haustorial hairs from epidermal cells, andthe development of densely staining cells at the haustorialapex. In addition, these changes occurred within a similar time-frameand sequence in root culture and radicle haustoria. Finally,root culture haustoria were found to be capable of attachingto sorghum host roots. Key words: Striga asiatica L., Kuntze, haustoria, root cultures, proteins, histology, 2D-PAGE     

Endogenous hormone levels and anatomical characters of haustoria in Santalum album L. seedlings before and after attachment to the host

The physiological and anatomical attributes of haustoria tissues in hemi-parasitic Santalum album L. seedlings, growing on the potential host, Kuhnia rosmarnifolia Vent., were investigated before and after attachment to the host. Quantization of endogenous levels of indole-3-acetic acid (IAA), zeatin (Z), zeatin riboside (ZR), GA-like substances (GAs) and abscisic acid (ABA) was performed by HPLC. Histological preparations were used to characterize structural differences between pre- and post-attachment haustoria. The contents of GAs and ABA were higher in attached haustoria, with 3.61 and 3.50μgg(-1) fresh weight, respectively, and three times higher than in non-attached haustoria. Cytokinins, Z, ZR and IAA levels were also high, and their contents in attached haustoria increased 2.04-, 2.17-, and 2.82-fold more, respectively, than in non-attached haustoria. A high auxin-to-cytokinin ratio contributed to haustorial development of S. album. A numerous amount of starch in parenchyma cells around the meristematic region above the haustorial gland and the endophyte tissue of the post-attachment haustoria were reported in a Santalaceae member for the first time. Many lysosomes were present and large-scale digestion of host cells occurred at the interface between the parasite and host. The haustorial penetration in S. album into the host stele was suggested to be a function of mechanical force and enzymatic activity. Analysis of the endogenous hormone levels and the structural characters in S. album haustoria indicated that the haustoria were able to synthesize phytohormones, which appeared to be necessary for cell division and differentiation during haustorial development. These results suggest that endogenous hormones are involved in the haustorial development of S. album and in water and nutrient transport in the host-parasite association.     

Progress in parasitic plant biology: host selection and nutrient transfer

Host range varies widely among species of parasitic plants. Parasitic plants realize host selection through induction by chemical molecular signals, including germination stimulants and haustoria-inducing factors (HIFs). Research on parasitic plant biology has provided information on germination, haustorium induction, invasion, and haustorial structures and functions. To date, some molecular mechanisms have been suggested to explain how germination stimulants work, involving a chemical change caused by addition of a nucleophilic protein receptor, and direct or indirect stimulation of ethylene generation. Haustorium initiation is induced by HIFs that are generated by HIF-releasing enzymes from the parasite or triggered by redox cycling between electrochemical states of the inducers. Haustorium attachment is non-specific, however, the attachment to a host is facilitated by mucilaginous substances produced by haustorial hairs. Following the attachment, the intrusive cells of parasites penetrate host cells or push their way through the host epidermis and cortex between host cells, and some types of cell wall-degrading enzymes may assist in the penetration process. After the establishment of host-parasite associations, parasitic plants develop special morphological structures (haustoria) and physiological characteristics, such as high transpiration rates, high leaf conductance, and low water potentials in hemiparasites, for nutrient transfer and resource acquisition from their hosts. Therefore, they negatively affect the growth and development and even cause death of their hosts.     

寄生药用植物管花肉苁蓉种子的离体萌发与吸器形成的形态学研究

研究了管花肉苁蓉(Cistanche tubulosa (Schenk) R. Wight)种子脱离寄主条件下的离体萌发和吸器形成的形态学,为其高产栽培提供参考。将经过层积处理的种子浸泡于赤霉素(1 000 mg.L-1)36小时,然后培养于1/2 MS(加500 mg.L-1酵母提取物、250 mg.L-1氯化钠和100 mg.L-1甘露醇,pH5.83)培养基上。培养两周后种子开始萌动,首先胚根从软化的内种皮的种孔端伸出,接着形成芽管状物,之后芽管状物逐渐伸长;17天后初生吸器开始发育,厚的带状表皮分化出若干吸根毛;在初生吸器顶端带状表皮围成窝状,表皮上的褶皱和窝内分布有乳头状突起。芽管状物延伸至4 mm就停止发育。吸根毛和乳突是黏着寄主根、刺穿寄主皮层发育为内生吸器最终建立寄生关系的基础。     

马铃薯对晚疫病水平抗性的组织细胞学观察

以马铃薯晚疫病水平抗性品种LBr-12和感病品种费乌瑞它为材料,采用普通光学和电子显微镜技术,系统研究了马铃薯与晚疫病菌(致病疫霉)互作的组织细胞学反应特征。观察结果显示:(1)接种后,水平抗性材料LBr-12出现过敏反应,病菌被限制在侵染点的几个细胞中,菌丝产生较少的分支和吸器。(2)感病品种费乌瑞它被侵染细胞呈蔓延趋势,菌丝产生较多的分支和吸器。(3)电镜观察发现,抗病品种上病菌的胞间菌丝、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,包括原生质的电子致密度加深、液泡增多变大、菌丝细胞壁不规则增厚、细胞器排列紊乱及解体、吸器母细胞及吸器形态异常、病菌最终畸形坏死,同时发现抗病品种受病菌侵染时可迅速产生结构防卫反应,形成的细胞壁沉积物使胞壁极度增厚或细胞膜上产生乳突状结构。     

OBSERVATIONS ON ROOT-PARASITISM IN CORDYLANTHUS (SCROPHULARIACEAE)

Eight species of Cordylanthus (Scrophulariaeeae: tribe Euphrasieae) representing the three major sections of the genus were successfully grown both with and without a host plant, demonstrating that the plants are facultative hemi-parasites capable of completing their life cycle without a host under the favorable conditions of greenhouse culture. The plants are almost certainly parasitic in the wild. Even though haustorial connections were formed by Cordylanthus on the roots of all host plants provided, most species were more vigorous when growing with certain hosts (Quercus, Pinus, Helianthus) than with others (Plantago, Phleum). This indicates a difference in the ability of the parasite to obtain materials successfully from the various plants used as hosts. Natural hosts are inferred for 15 species of Cordylanthus from the presence of associated species in nature. The haustoria are exogenous in origin, being formed by dedifferentiation of several cells in the hypo-dermal or subhypodermal layers of the rootlets. Anatomically the haustoria differ from those reported for other members of Euphrasieae in having secondary vascular structure and abundant connecting strands. The vascular structure of haustoria is composed mainly of specialized vessel elements. Root-parasitism gives Cordylanthus the ability to grow in arid areas during the summer after most other annuals have died, thus successfully equipping the plants for survival and expansion in western North America.     

寄生药用植物管花肉苁蓉种子的离体萌发与吸器形成的形态学研究

研究了管花肉苁蓉(Cistanche tubulosa(Schenk)R.Wight)种子脱离寄主条件下的离体萌发和吸器形成的形态学,为其高产栽培提供参考.将经过层积处理的种子浸泡于赤霉素(1000 mg·L-1)36小时,然后培养于1/2 MS(加500mg·L-1酵母提取物、250 mg·L-1氯化钠和100mg·L-1甘露醇,pH5.83)培养基上.培养两周后种子开始萌动,首先胚根从软化的内种皮的种孔端伸出,接着形成芽管状物,之后芽管状物逐渐伸长;17天后初生吸器开始发育,厚的带状表皮分化出若干吸根毛;在初生吸器顶端带状表皮围成窝状,表皮上的褶皱和窝内分布有乳头状突起.芽管状物延伸至4mm就停止发育.吸根毛和乳突是黏着寄主根、刺穿寄主皮层发育为内生吸器最终建立寄生关系的基础.     

Ultrastructure of the host-pathogen interface in daylily leaves infected by the rust fungus Puccinia hemerocallidis

Summary. Transmission electron microscopy was used to examine details of the host–pathogen interface in daylily leaf cells infected by the rust fungus Puccinia hemerocallidis. Samples were prepared for study by high-pressure freezing followed by freeze substitution. The outstanding preservation of ultrastructural details afforded by this fixation protocol greatly facilitated the study of this host–pathogen interface. The extrahaustorial membrane that separated each dikaryotic haustorium from the cytoplasm of its host cell was especially well preserved and appeared almost completely smooth in profile. Large aggregations of tubular cytoplasmic elements were present near haustoria in infected host cells. Many of these tubular elements were found to be continuous with the extrahaustorial membrane and conspicuous electron-dense deposits present in the extrahaustorial matrix extended into these elements. The use of gold-conjugated wheat germ agglutinin for labeling of chitin revealed that these deposits were not part of the haustorial wall. Portions of many of the tubular elements associated with haustoria were conspicuously beaded in appearance. Some tubular elements were found to be continuous with flattened cisternae that in turn bore short beaded chains. Distinctive tubular-vesicular complexes previously reported only in cryofixed rust haustoria also were found in the haustoria of P. hemerocallidis. Received July 6, 2001 Accepted October 3, 2001     

Host selectivity,haustorial anatomy and impact of the invasive parasite Parentucellia viscosa on floodplain vegetative communities in Japan

The parasitic plant Parentucellia viscosa has been introduced recently to the Japanese floodplain. Because of its parasitic nature and high fecundity, P. viscosa may well become a major plant invader with a significant impact on floodplain ecosystems. Thus, a knowledge of the host selectivity of P. viscosa will not only provide basic biological information, but will also contribute to the understanding of floodplain vegetation conservation. We evaluated the host selectivity by comparing the observed numbers of haustoria with those expected from the relative below‐ground biomass. In addition, we examined the haustorial anatomy to determine whether haustoria are functional, and compared the above‐ground biomass of three functional plant groups (grasses, legumes and nonleguminous forbs) in intact and parasite removal quadrats. We found that haustoria were nonrandomly distributed amongst host species, suggesting host preference for Poaceae and Fabaceae. In addition, haustoria attached to certain forbs did not penetrate into the stele. The above‐ground biomass of grasses and legumes was increased significantly by the removal of P. viscosa, but the biomass of forbs did not change significantly. These results suggest that host preference depresses the performance of Poaceae and Fabaceae, thus affecting the competitive relationships among plants, meaning that P. viscosa may pose a serious threat to indigenous endangered legumes and grasses. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 170 , 69–78.     

Interface between haustoria of parasitic members of the Scrophulariaceae and their hosts: A histochemical and immunocytochemical approach

Summary The haustorial structure of three African parasitic members of the family Scrophulariaceae (Buchnera hispida, Rhamphicarpa fistulosa, andStriga hermonthica) has been studied with regard to the interface between haustoria and the invaded host roots. Immunocytochemical observations at the light and electron microscopical level were carried out with monoclonal antibodies against pectin. JIM5, JIM7, and hydroxyproline-rich glycoprotein (HRGP), LM1. Lignins have been visualized by phloroglucinolhydrochloric acid staining. At the margin of the lateral interface (contact area of host root cortex and parasite cells), JIM5- and JIM7-labelled substances accumulate between parasite papillae and the host root surface indicating that pectins are implicated in sealing the parasite to the attacked host organ. The lateral interface is characterized by the presence of compressed, necrotic host cells, whereas the central interface (contact area between host stele and parasite cells) is generally devoid of host cell remnants. Phenolic substances and/or lignins can be found at the site of penetration of the haustorium into the host root. These observations and the fact that HRGPs accumulate at the host side of the interface support the view of, at least, a partial defense reaction in the invaded host root tissues. Within haustoria, HRGPs were restricted to differentiating xylem elements, implying a spatio-temporal regulation of HRGPs in developmental processes.Abbreviations BSA bovine serum albumin - FITC fluorescein isothiocyanate - HRGP hydroxyproline-rich glycoprotein - LM light microscopy - MAb monoclonal antibody - TBSB Tris-buffered saline with bovine serum albumin - TBSB-T Tris-buffered saline with bovine serum albumin and Tween 20 - TEM transmission electron microscopy     

THE HOST-PARASITE INTERFACE OF ALBUGO CANDIDA ON RAPHANUS SATIVUS

The fine structure of the intercellular hyphae of the obligate parasite Albugo candida infecting radish does not differ markedly from that described previously for cells of Peronospora manshurica. The stalked, capitate haustoria do not contain nuclei and are packed with mitochondria and lomasomes. The fungal plasma membrane and cell wall are continuous from the intercellular hypha throughout the haustorium except that there is no evidence of fungal cell wall around a portion of the haustorial stalk proximal to the haustorial head. Within the vacuolate host mesophyll cell, the haustorium is always surrounded by host plasma membrane and with at least a thin layer of host cytoplasm. The host cell wall invaginates at the point of haustorial penetration to form a short sheath around the region of penetration, but normally there is no host cell wall around the balance of the haustorium. About 1% of the haustoria observed were necrotic, and these were invariably walled-off completely from host cytoplasm by host cell wall. An amorphous, moderately electron-dense encapsulation lies between the haustorium proper and the host plasma membrane and extends into the penetration region between the sheath and the fungal cell wall. Invaded host cells contain more ribosomal-rich ground cytoplasm than uninfected cells. Glandular-like systems of tubules and connecting vesicles are often numerous in host cytoplasm in the vicinity of haustorial heads. These tubules open into the encapsulation, their limiting unit membranes being continuous with the host plasma membrane. We suggest that these represent a secretory mechanism of the host specifically induced by the parasite.     

寄生植物无根藤吸器发育过程中酸性磷酸酯酶与细胞分裂素变化的研究

无根藤（ＣａｓｓｙｔｈａｆｉｌｉｆｏｒｍｉｓＬ．）吸器发育过程中,细胞内酸性磷酸酯酶与细胞分裂素含量表现出动态变化。吸器发生时,其发生部位的异成烯基腺昔（ｉＰＡ）与玉米素核昔（ＺＲ）含量较对照部位分别高２０倍和６倍,表明吸器的发育与细胞分裂素密切相关。吸器的侵入生长是由于酸性磷酸酯酶影响寄主组织细胞,然后通过吸器生长的压力挤碎寄主细胞而侵入生长,是化学作用与物理作用共同作用的结果。     

Suppression of the powdery mildew pathogen by chitinase microinjected into barley coleoptile epidermal cells

An exogenous chitinase from Streptomyces griseus was introduced into coleoptile epidermal cells of barley (Hordeum vulgare) by microinjection, and the effect of injected chitinase on the growth or development of the powdery mildew pathogen (Erysiphe graminis f. sp. hordei) was examined. Prior to microinjection, an enzymatic degradation of fungal haustorium, the organ taking nutrients from host plant cells, was examined by treating fixed coleoptile epidermis harboring haustoria with this enzyme. The result showed that haustoria were effectively digested by chitinase, suggesting the effectiveness of chitinase treatment for suppressing the fungal development. Microinjection of chitinase was conducted using living coleoptile tissues inoculated with the pathogen. Epidermal cells in which the haustorial primordia had been formed, or in which the haustoria had matured, were selected as targets for injection. The result clearly indicated that injection at the stage of primordium formation was effective in completely digesting haustoria and suppressing the subsequent formation of secondary hyphae of the pathogen. In microinjection after haustorial maturation, hyphal elongation was considerably suppressed though there was no detectable morphological change in the haustoria. Thus, the present study provides the experimental basis for genetically manipulating barley to produce transgenic plants resistant to the powdery mildew disease.     

Structure and Development of the Primary Haustorium in Alectra vogelii Benth. (Scrophulariaceae)

Anatomical observations were made on the structure and developmentof the primary haustorium of Alectra vogelii. Its developmentinvolves a mutual aggressive growth of both the host and parasitetissues resulting in the formation of a very large and complextuberous organ. One of the host tissues whose growth is stimulatedby parasite infection is the pericycle whose cells divide repeatedlyand grow around and within the parasite haustorial cortex. Fromvarious points of the proliferating host pericycle, roots becomeinitiated and eventually the entire surface of the haustoriumbecomes covered with these roots. We have referred to them as‘haustorial roots’, a term which we have re-examinedand redefined. True xylary connections are established not onlybetween the parasite and the host root but also between theparasite and these ‘haustorial roots’. The uniquedevelopment of primary haustorium and ‘haustorial roots’in A. vogelii is discussed in relation to the development ofprimary haustoria in other root parasites.     

梨胶锈菌性孢子和锈孢子阶段吸器的超微结构研究

本文对梨胶锈菌性子期和锈子期菌丝吸器的形成方式、吸器及其与寄主细胞界面的超微结构进行了研究。观察结果表明：梨胶锈菌性子期和锈子期寄主胞间菌丝吸器的形成方式有两种：一种是由寄主胞间菌丝直接形成吸器;另一种是由寄主胞间菌丝先形成吸器母细胞,然后由吸器母细胞形成吸器。吸器在开始形成时只是一个乳头状的侵入楔,以后逐渐形成囊状、镰刀状、指状及其它不规则形状的吸器。多数吸器分化为颈和吸器主体两部分,在颈部及部分吸器主体外有一个由类似寄主细胞壁物质形成的领圈。吸器内部的超微结构与寄主胞间菌丝基本相同,但吸器壁比胞间菌丝或吸器母细胞的壁薄。吸器鞘的厚度随着吸器伸长膨大 而逐渐增厚。