Monoclonal antibodies to specific components of the Dutch elm disease pathogen Ophiostoma ulmi

The potential of polyclonal antisera and monoclonal antibodies to differentiate the EAN and NAN aggressive subgroups of Ophiostoma ulmi was explored. Polyclonal antisera, when tested by ELISA, cross-reacted widely with unrelated species and failed to distinguish between the two aggressive subgroups but small quantitative differences were found, particularly between antigens secreted overnight, by EAN and NAN germlings. Monoclonal antibodies were raised in mice against mycelial homogenates. From two fusions, 33 cell lines were raised that secreted antibodies positive for O. ulmi. Approximately one third were non-specific; 11 were specific either to species or subspecies. Two cell lines differentiated mycelial antigens of the aggressive isolates of O. ulmi from those of the non-aggressive subgroup, but not antigens from surface washings. Only quantitative differences were detected between the EAN and NAN aggressive subgroups. Almost all the monoclonal antibodies and antiserum recognized antigens present in surface washings of cultures on solid medium, in cell-free extracts of mycelial homogenates, in cell-free culture fluids, and in substances secreted overnight by germinating spores. Specific detection of such molecules promises to provide a highly sensitive mechanism for studying early pathogen/host plant interactions. Most of the monoclonal antibodies appeared to have potential diagnostic value; they gave readings twofold to tenfold higher with extracts from diseased than from healthy tissue. However, one cell line that secreted antibodies specific to O. ulmi cross-reacted strongly with extracts of healthy tissue.     

Influence of the d2 factor on survival and infection by the Dutch elm disease pathogen Ophiostoma ulmi

A particular form of mycovirus, the d² factor, which attacks the elm pathogen Ophiostoma ulmi, was found to be extremely deleterious to this fungus both in vitro and in vivo. Most significantly, d²-infected conidia were poor at persisting in feeding grooves, the usual infection court of the pathogen, and also showed a 50-fold reduction in successful xylem infection compared with their healthy counterparts. In addition, transmission of the d² factor from diseased to healthy isolates of O. ulmi was found to take place in feeding grooves, indicating that the fungus exists in a mycelial phase prior to invading xylem vessels.     

Detection of corn stunt spiroplasma in vivo by ELISA using antisera to extracts from infected corn plants (Zea mays)

Antisera were raised in rabbits to extracts from healthy corn plants and from plants infected with corn stunt spiroplasma (CSS); they were prepared by partially purifying sap or tissue homogenates from whole stems by differential centrifugation and filtration. The titres of CSS-specific antibody in the antisera to diseased plant extracts (DPE) were monitored by spiroplasma deformation tests. After cross-absorption against healthy plant extracts, the DPE antisera detected cultured CSS at concentrations exceeding 10⁶ cells/ml, compared with less than 10⁵ cells/ml for homologous antiserum, and reacted more strongly against extracts from diseased than from healthy plants. A comparison of techniques for extracting diseased plant tissues showed that the reaction of stems and midribs against cultured CSS antiserum was two to three times stronger if the samples were lyophilized before extraction. When extracts from lyophilized tissues were used as test antigens the DPE antisera discriminated between diseased and healthy corn stems, midribs and roots as effectively as antiserum to cultured CSS. Preliminary attempts to detect CSS in its vector, Dalbulus maidis , were complicated by non-specific reactions of insect tissue extracts but concentrations equivalent to at least one CSS-infected insect/ml of sample were detected by antiserum to cultured CSS.     

Transmission of double-stranded RNA and a disease factor in Ophiostoma ulmi

A diseased isolate of Ophiostoma ulmi was found to contain 10 segments of double-stranded RNA (dsRNA) with molecular weights ranging from 2.40 x 10⁶ to 0.23 x 10⁶. In contrast seven other healthy isolates in the same vegetative compatibility group contained either no dsRNA or up to four dsRNA segments. Transmission of the disease to healthy isolates by hyphal anastomosis was accompanied by-transmission of the 10 dsRNA segments. In a genetic cross in which the diseased isolate acted as the female parent single-ascospore progeny were healthy and either contained no dsRNA or only one segment of dsRNA. When elm trees were inoculated with diseased isolates, subsequent reisolations were healthy and retained only two to seven of the dsRNA segments or were diseased and retained all 10 dsRNA segments. Following conidiogenesis a diseased isolate gave rise to single-conidial progeny which were either slow growing and diseased, like their parent, with all 10 dsRNA segments, or faster growing like healthy isolates. Some of the faster growing conidial isolates retained only two to seven of the dsRNA segments and were disease-free. However a majority of the faster growing conidial isolates retained all 10 dsRN A segments and were shown to carry the disease in a latent form. The possibility that the disease of O. ulmi is conferred by specific segments of dsRNA and the potential of d-factors for the control of Dutch elm disease are discussed.     

Development of a monoclonal antibody immunoassay for the eyespot pathogen Pseudocercosporella herpotrichoides

A double-antibody-sandwich ELISA test has been developed for the detection of Pseudocercosporella herpotrichoides using a highly specific monoclonal antibody PH-10 as the capture antibody and genus-specific rabbit polyclonal antiserum as the detector antibody. The assay recognizes extracts from plants both artificially and naturally infected with P. herpotrichoides giving at least three-fold higher absorbance values with extracts from Pseudocercosporella-infected tissue than with extracts from healthy tissues or from tissues naturally infected with Microdochium nivale, Rhizoctonia cerealis or material artificially inoculated with P. anguioides. The assay tested positively against all isolates of P. herpotrichoides , including both W-type and R-type isolates. In this assay system, extraction of the antigen from the stem bases of infected plants is a one-step process not requiring any dilution procedures. The assay can be used to detect the pathogen in presymptomatic infected seedlings. The immunogen used to generate the specific monoclonal antibody and the rabbit antiserum was a mycelial extract from which the high-molecular-weight proteins and glycoproteins had been removed by ammonium sulphate precipitation. The high-molecular-weight fraction was shown to contain cross-reactive antigens; it induced antiserum in mice that cross-reacted with the other stem-base fungi even at high dilutions. The monoclonal antibody PH-10 is an IgM antibody. Heat and periodate treatment of the antigen indicate that it is a glycoprotein and that the epitope recognized by the antibody is a protein.     

Development of a monoclonal antibody-based immunodetection assay for Botrytis cinerea

Three hybridoma cell lines secreting antibodies that specifically recognize Botrytis cinerea and B.fabae , but not B. allii, have been raised from splenocytes of mice immunized with a low molecular-weight fraction (30 kDa) from surface washings of B. cinerea. Antibodies from these cell lines have been used to develop an antigen-based elisa test that will detect B. cinerea in strawberries. This monoclonal antibody immunoassay detection assay should prove useful to both the cut-flower and wine industries. Supernatants from the three specific cell lines recognize mycelial fragments, saline extracts of mycelia and germinating conidia by both ELISA and immunofluorescence. Recognition of non-germinating spores is poor. Supernatants from the specific cell lines did not recognize other fungi normally involved in post-harvest spoilage of fruits and vegetables. Supernatants from KH4 gave the lowest background values with healthy tissue. Indirect evidence from heat, protease and periodate treatment of the antigens indicates that antibodies from all three specific cell lines are recognizing carbohydrate epitopes on a glycoprotein.     

一种高效分离荔枝霜疫霉病菌的新方法

作者将常规植物病原菌组织分离法改进成一种适合于荔枝霜疫霉病菌分离的简便、高效的新方法。分离的荔枝果实经过100 μg/mL多菌灵及36 μg/mL链霉素的混合药液浸泡处理,在无菌保湿的条件下采用病果对健果进行病害诱发后进行病原菌的分离纯化。该新方法分离荔枝霜疫霉病菌的成功几率大于传统组织分离法。     

Detection methods for Xanithomonas campestris pv. graminis on forage grasses1

In Norway seven different grass species were found to be attacked by Xanthomonas campestris pv. graminis (bacterial wilt of forage grasses). Among these, Phleum pratense is the most susceptible and common host. Considerable damage may occur after long periods of hot and dry weather, while in cool and wet periods hardly any diseased plants are found. The pathogen may be identified by isolation and biochemical tests. Specific antibodies used in immunofluorescence and ELISA had a high degree of sensitivity and specificity against the target bacterium. The two methods were used for screening pure cultures and detecting bacteria directly in plant tissue extracts. Their application revealed the presence of low numbers of bacteria in symptomless plants and a discontinuous distribution within the plant.     

Differences in cerato-ulmin production between the EAN, NAN and non-aggressive subgroups of Ophiostoma ulmi

A test of comparative in vitro cerato-ulmin wilt toxin production in the aggressive and non-aggressive subgroups of the Dutch elm disease pathogen Ophiostoma ulmi was carried out by turbidity and ELISA methods. Ten non-aggressive, ten EAN aggressive and ten NAN aggressive isolates were tested from a range of geographical sources. In liquid shake cultures the non-aggressive isolates produced the greatest and the NAN aggressives the least mean biomass. Despite considerable variation in cerato-ulmin production by individual isolates in three separate experiments, both the turbidity and ELISA methods showed a clear separation of the non-aggressive and aggressive subgroups. Non-aggressive isolates produced little or no cerato-ulmin (ELISA range of means 0–56.0 ng/ml) and EAN and NAN aggressive isolates moderate to high levels (EAN 1.6–89.0 × 10⁴ ng/ml and NAN 0.2–300 × 10⁴ ng/ml). In the aggressive isolates no correlation was detected between cerato-ulmin production and either biomass or pathogenicity to clonal Commelin elm. The role of cerato-ulmin in the pathogenicity of O. ulmi is discussed.     

Development of crown and root rot disease of tomato under irrigation with saline water

ABSTRACT We studied the effect of water salinity on the incidence and severity of crown and root rot disease of tomato, as well as on the pathogen and on the plant's response to the pathogen. Irrigation with saline water significantly increased disease severity in tomato transplants inoculated with Fusarium oxysporum f. sp. radicis-lycopersici, and mineral fertilization further increased it. In one field experiment, disease incidence in plots irrigated with saline water (electrical conductivity [EC] = 3.2 +/- 0.1 dS m(-1)) and in those irrigated with fresh water (EC = 0.4 +/- 0.1 dS m(-1)) was 75 and 38%, respectively. Disease onset was earlier and yield was lower in plots irrigated with saline water. In a second field experiment, final disease incidence 250 days after planting, was 12% in plants which had been irrigated with saline water (EC = 4.6 +/- 0.1 dS m(-1)) and 4% in those irrigated with fresh water (EC = 1.2 +/- 0.1 dS m(-1)). Irrigation of tomato transplants with 20 mM NaCl did not inhibit plant development, but partial inhibition was observed at higher NaCl concentrations. Growth of the pathogen in culture or survival of conidia added to soil were not affected by saline water. Plants which were preirrigated with saline water were more severely diseased than those preirrigated with tap water. It was concluded that disease increases effected by saline water are associated with the latter's effect on plant response.     

Sensitivity of different methods for the detection of Ralstonia solanacearum in potato tuber extracts

The sensitivities of various methods for the detection of Ralstonia solanacearum following dilution in healthy potato tuber tissue macerate were compared. Estimated pathogen populations in undiluted macerates, from samples of 200 heel-end vascular cores each containing a single diseased and 199 healthy tubers, ranged from 1.2 × 10⁶–7.4 × 10⁷ colony-forming units per ml. Following concentration by high-speed centrifugation and resuspension in phosphate buffer, the pathogen was detected by all methods studied, including culture on semi-selective media, enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescent-antibody staining (IFAS) of fixed cells, immunofluorescent colony staining (IFCS), detection of specific DNA sequences following amplification by the polymerase chain reaction (PCR) and bioassay in tomato seedlings. Both ELISA and PCR methods were improved by pre-enrichment of samples in semi-selective broth prior to testing. A nested PCR method was evaluated which could detect fewer than 10 cells per ml in the potato extracts. Of the other methods only dilution plating on semi-selective medium and tomato bioassay could detect fewer than 10⁴ cells per ml. In order to combine ease and speed of use with sensitive detection, it was recommended that a series of methods be used for routine screening of potato tuber stocks for infection by R. solanacearum.     

应用伪钝绥螨防治苹果全爪螨的试验

1987～1988年田间应用试验表明,青岛地区生态条件可以满足伪钝绥螨生存和发展的要求.5月下或6月初,以1:50的益害比,释放伪钝绥螨,可控制苹果全爪螨为害,减少果园施用杀螨剂3次。伪钝绥螨在当地可以自然越冬,完成周年循环,建立种群,成为控制叶螨发生的有效天敌。     

南疆红枣黑斑病病原鉴定及其保守基因序列分析

正红枣黑斑病(jujube black spot)又称"烂果病"、"黑头病",在新疆南疆地区发生严重。2013—2014年阿克苏、和田地区红枣黑斑病发病率达20%~30%,重病枣园发病率高达40%~50%。红枣黑斑病严重制约着新疆红枣产业的健康发展。目前,国内在红枣黑斑病病原认知上仍存在较大分歧,报道的病原主要为链格孢(Alternaria alternata)~([1~3]),也有细极链格孢(Alternaria tenuissima)危害的报道~([4])。本文通过对南疆红枣黑斑病菌形态特征研究和田间病原回接试验,结合分子生物学手     

Development of a polyclonal antibody-based immunoassay for the early detection of Sclerotinia sclerotiorum in rapeseed petals

A serological test has been developed that allows the early detection of infection of young petals by Sclerotinia sclerotiorum , an important pathogen of rapeseed. Two steps were required to obtain an antiserum sufficiently specific for S. sclerotiorum. Soluble mycelial extracts of S. sclerotiorum were used to produce the first generation polyclonal antiserum. This was not specific for S. sclerotiorum in double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and allowed the screening of cross-reacting fungal species such as Botrytis cinerea , a pathogen commonly present on rapeseed petals. Using a polyclonal anti- B. cinerea serum enabled the absorption, by serial cycles, of S. sclerotiorum antigens common to B. cinerea. Residual antigens were then used as immunogens for the production of two second generation antisera (S1 and S2) which were then tested by DAS-ELISA. Cross-reactions with B. cinerea decreased with purification cycles of the immunogen whereas Cross-reactions with some unrelated fungi slightly increased. S. sclerotiorum and B. cinerea were distinguishable using antiserum S2.     

柑桔溃疡病苗化学治疗试验

对柑桔溃疡病苗化学治疗试验结果指出:病苗在1000～2000单位/毫升链霉素+1:100酒精的混合药液中浸1～3小时,便能把病苗治好。处理过的果苗,生长仍然良好。病苗在1:1000～1500的升汞水+1:100酒精的混合药液中1～3小时,也有同样的治疗效果,但苗木生长略差,死亡率略高。用链霉素、升汞水或波尔多液喷射经剪掉病枝叶,或不剪掉病枝叶的病苗,均不能把病苗完全肃清。对柑桔溃疡病的防治,许多国家(如南非、美国等),认为只有毁灭病株,才有可能彻底消灭病原。有时病菌能潜伏数年,如南非有一果园在1918年毁灭病株,1924年重种,当年又发生病害。因此,毁灭病株后,需要连续数年检查,证实无病,才能复种。     

Maize seed chitinase is modified by a protein secreted by Bipolaris zeicola

Plants contain defense mechanisms that prevent infection by most fungi. Some specialized fungi have the ability to overcome plant defenses. The Zea mays (maize) seed chitinase ChitA has been previously reported as an antifungal protein. Here we report that ChitA is converted to a modified form by a protein secreted by the ear-rot pathogen Bipolaris zeicola (Holomorph = Cochliobolus carbonum). In denaturing chitinase zymograms, ChitA activity was detected in protein extracts from healthy seeds, but not in those from diseased seeds of ears that had been inoculated with B. zeicola. Furthermore, addition of proteins from diseased seeds to those from healthy seeds resulted in the loss of detectable ChitA activity. This indicated that protein extracts from diseased seeds contained a molecule that targets ChitA. We determined that this molecule is a secreted protein we term B. zeicola chitinase modifying protein (Bz-cmp). We purified ChitA from maize seed and Bz-cmp from sterile cultures of B. zeicola. Biochemical experiments demonstrated that Bz-cmp catalytically converts ChitA into a modified form that has reduced molecular mass in SDS-PAGE gels. Native chitinase zymogram analysis of modified and unmodified ChitA demonstrated that, contrary to our initial observation, Bz-cmp does not impair ChitA's chitinase activity but rather its chitin-binding ability. Our findings suggest that ChitA is an important component of defense in developing maize seeds and identifies Bz-cmp as a potential contributor to pathogenicity.     

Antibody production and identity of MLOs associated with sugar-cane whiteleaf disease and bermuda-grass whiteleaf disease from Thailand

Cuttings from sugar-cane plants with symptoms of whiteleaf disease and bermuda-grass plants also showing whiteleaf symptoms were collected in Thailand and transferred to a glasshouse at East Mailing. Using these plants as source material, procedures were devised for the partial purification of MLO-associated immunogens. Antisera were raised separately to the sugar-cane whiteleaf and bermuda-grass whiteleaf immunogens. These antisera exhibited specificity for their homologous MLO antigens in F(ab')₂ indirect enzyme immunosorbent assays (ELISA). Detection of MLO infection was possible in crude tissue extracts diluted several hundredfold. No cross-reactions were observed in reciprocal tests between these two MLO sources and their antisera, nor were there reactions between either antiserum and extracts of other graminaceous hosts showing symptoms of natural whiteleaf infection. No cross-reactions were obtained in reciprocal tests with Spiroplasma citri or aster yellows MLO and their homologous antibodies.     

Fine structure of phloematic trypanosomatid–coconut tree interaction

Phytomonas wilt or Hartrot is a fatal disease of palm (Arecaceae) species including Cocos nucifera (coconut) and is caused by a phloematic trypanosomatid, a promastigote parasite that inhabits phloem sieve elements of disease palms. In the present work, we described the morphology of the interaction between a phloematic trypanosomatid (Phytomonas staheli) and C. nucifera. Two varieties and one ecotype of the adult coconut palm from northeast and southeast Brazil were analyzed, totaling 34,000 plants. Coconut palm losses due to Hartrot varied according to the variety or ecotype and geographic area. Occurrence of Hartrot was insignificant in Rio de Janeiro state (southeast), but in Bahia state (northeast) losses were substantial when appropriate cultural practices were not applied. Symptomatic and healthy palm tissues were analyzed by light and electron microscopy. Laboratory diagnoses revealed the twisted promastigote form of phloematic trypanosomatids in the extracts of shoot apex, leaves, stems and inflorescence in diseased plants, but not in the healthy ones. No parasites were found in the roots. Although the general anatomy of healthy and diseased palms was similar, callose deposition in the sieve plates was revealed by histochemistry and immunocytochemistry in the diseased tissue. Plugging by the P-protein and plastid alterations was also observed. Our observations strongly suggest that parasite traffic between sieve elements took place, although their cell bodies were larger than the sieve pores. Phloematic trypanosomatid proliferation in the sieve tube elements might interrupt the transport of phloem or/and consume plant nutrients. In addition, an association between the percentage of sieve elements colonized by pathogen in palm tissues and disease severity was established.     

Biology and integrated control of Pestalotiopsis on container-grown ericaceous crops

Pestalotiopsis isolates obtained from the foliage, stem-base and roots of diseased container-grown ericaceous crops (Calluna, Erica, Pieris and Rhododendron) collected from UK nurseries were identified as Pestalotiopsis sydowiana (Bresad) B Sutton on the basis of conidia morphology. Inoculum sources of the pathogen included diseased stock plants, crop debris, nursery soils, used growing media, pots and floor covering, and dust collected from greenhouse walkways. Isolates were not host-specific and infected other species of ericaceous plants, with typical symptoms including browning of foliage, stems and roots, and the presence of black or greenish black acervuli on diseased tissue. The optimum temperature for growth of three selected isolates of the pathogen was 20-25 degrees C, with little or no growth occurring below 5 or above 30 degrees C. Growth occurred over pH 2.6-8.6, with optimum at 5.5. Decreases in matric potential from -0.3 to -4.0 MPa reduced growth, which was totally inhibited at -6.5 MPa. Greenhouse trials were conducted to evaluate the effects of disease management methods (irrigation, flooring/pot disinfection and fungicide application) on control of the pathogen on potted plants of C vulgaris. Disease incidence and foliar browning caused by P sydowiana were less on fungicide-treated (five-spray programme of alternating prochloraz and carbendazim) potted plants watered by sub-irrigation compared with watering from overhead. Single and combined treatments of flooring/pot disinfection (hydrogen peroxide/peracetic acid) and the five-spray fungicide programme significantly reduced disease incidence and severity compared with dipping pots in water. The combined disinfection and fungicide programme significantly reduced disease incidence and severity, compared to disinfection or fungicide application alone. The importance of these findings for the integrated control of P sydowiana on ericaceous plant nurseries is discussed.     

华重楼斑枯病病原鉴定

 2018年,四川汶川县重楼种植区的华重楼出现一种新的病害—华重楼斑枯病,该病害主要危害叶片,发生率为35%,天气适宜时可引起整株叶片枯死。为鉴定引起四川汶川县华重楼斑枯病的病原菌,本试验采用组织分离法对病原菌进行分离,利用科赫氏法则验证其致病性,依据菌株的形态学和基于rDNA-ITS和RPB2基因序列分析对病原菌进行鉴定。结果表明,分离菌株的菌落形态、分生孢子器和分生孢子形态与Didymella sp.相似;经分子生物学鉴定,该菌ITS-RPB2基因序列与亚隔孢壳属D. glomerata(登录号为FJ427013、GU371781)的同源性为100%。因此,将引起重楼斑枯病的病原菌鉴定为D. glomerata。