Entamoeba histolytica: surface proteolytic activity and its relationship with in vitro virulence.

We determined the surface-associated proteolytic activity in three Entamoeba histolytica Schaudinn, 1903 strains (monoxenic HM1, axenic HM1, and HK9) of known virulence and its relationship with collagenase activity. Both activities were also determined in axenic HM1 amoebae trophozoites which were sensitive and resistant to complement-mediated lysis. Surface proteolytic activity was determined in glutaraldehyde-fixed E. histolytica trophozoites, which degraded the insoluble substrate, hide powder azure, and cleaved the human immunoglobulin G heavy chain in a time-dependent fashion, at neutral pH, in presence of 2-mercaptoethanol as cysteine protease activator. Surface proteolytic activity was strain dependent: monoxenic HM1 > axenic HM1 > axenic HK9. This activity correlated with collagenolytic activity (p < 0.05). Acquisition of resistance to complement-mediated lysis by axenic HM1 strain did not modify either surface proteases or collagenase expression. Our results suggest that this surface proteolytic activity could be used as an in vitro virulence marker for E. histolytica.     

Genotypic and Pathotypic Diversity in Xanthomonas oryzae pv. oryzae in Nepal

ABSTRACT Among the 171 strains of Xanthomonas oryzae pv. oryzae (the bacterial blight pathogen of rice) collected from eight rice-producing zones in Nepal, 31 molecular haplotypes were distinguished using two polymerase chain reaction-based assays. Six common haplotypes represented nearly 63% of the strains, and some haplotypes were geographically dispersed. Multiple correspondence analysis divided the collection into five putative genetic lineages. Lineages 1, 2, and 4 were the most frequently detected and occurred in diverse geographic populations. Twenty-six pathotypes (virulence phenotypes) of X. oryzae pv. oryzae were identified using 11 near-iso-genic rice lines, each containing a single gene for resistance. The 26 pathotypes grouped into five clusters, and cluster 1 contained wide virulence spectrum strains from all geographic populations. Although molecular variation was greatest between strains of different virulence phenotypes, some variation was observed among strains with identical virulence. There was a weak correlation (r = 0.52) between molecular haplotypes and virulence phenotypes. There are two major groups of X. oryzae pv. oryzae in Nepal. One group consists of strains with high molecular polymorphism and many pathotypes that are either virulent to the 11 major resistance genes or avirulent only to Xa21. Strains in the second group have low molecular polymorphism and are avirulent to Xa4, xa5, Xa7, and Xa21.     

Analysis of soluble Entamoeba histolytica antigen using enzyme-L inked electroimmunotransfer blots

Soluble antigens of ten strains of E. histolytica were studied by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and enzyme-linked electroimmunotransfer blots (EITB). No relations of immune replicas to virulence, geographical origin and method of cultivation (xenic or axenic culture) were found. Antigens of all ten strains tested precipitated with anti-E. histolytica human serum in the area of 30-43 kD. Antigen of HK-9 strain created in this area a characteristic pattern with all sera containing the specific anti-E. histolytica antibodies and, therefore, EITB can be used for excluding false positive results in ELISA.     

Effect of geraniol,a plant-based alcohol monoterpene oil,against <Emphasis Type="Italic">Meloidogyne javanica</Emphasis>

In continuing previous work on the role of AAA+ proteins of the apple proliferation agent ‘Candidatus Phytoplasma mali’ in phytoplasma virulence and suppression of virulence, 147 full-length deduced protein sequences of AAA+ ATPase AP460 of single- and multiple-strain accessions were examined. This approach revealed that in this protein two regions can be distinguished. Region 1, located in the N-terminal part, is characterized by 14 highly conserved substitutions associated with the suppression of virulence. However, these substitutions were not present in all attenuated strains. In the more diverse region 2, located in the C-terminal part, highly conserved substitutions associated with two groups of virulence were identified. In addition to the virulence-related residues, three other groups of conserved substitutions are present in strains with attenuated virulence with or without the presence of suppression-associated substitutions in region 1. In one of these groups, substitutions next to key residues of the ATPase motifs sensor 1 and 2 and arginine finger do occur that seem to affect ATPase function. This group of substitution was present in all attenuated strains. From these findings it can be concluded that two different mechanisms of suppression exist of which the supposed effect on ATPase function seems to be more important than the suppression-associated substitutions in region 1. The presence of virulent, avirulent and suppressive strains in a tree leads in the commonly occurring multiple infections to interactions in which the resulting virulence is determined by the numerical relation of virulent and attenuated strains.     

Distribution of Xanthomonas oryzae pv. oryzae Strains Virulent to Xa21 in Korea

ABSTRACT Strains of Xanthomonas oryzae pv. oryzae that are virulent to rice lines carrying the Xa21 resistance gene were widely distributed in Korea. A total of 105 strains collected during 1987 to 1996 in Korea was characterized by pathogenicity tests and restriction fragment length polymorphism analysis of the XorII methyltransferase (xorIIM) and avrXa10 genes. Although the lesion lengths on rice line IRBB21, which carries Xa21, decreased as plant age increased, resistance and susceptibility of the plants to 31 strains were clearly differentiated at the seedling (14, 21, and 28 days old), maximum tillering, and flag leaf stages. The resistance or susceptibility of seedlings was correlated with bacterial populations within an inoculated leaf. There was a significant change in the population structure of X. oryzae pv. oryzae with regard to virulence to Xa21 over the last 10 years; this change in population was confirmed by genome analysis. Lineage I, which is avirulent to Xa21 and does not have a genomic xorIIM homolog, was the predominant lineage found between 1987 and 1989, while lineage II, which is virulent to Xa21 and contains the xorIIM homolog, was predominant in strains collected between 1994 and 1995. Our results demonstrate that introduction of Xa21 into commercial rice should be based on the regional structure of X. oryzae pv. oryzae populations and suggest that Xa21 will not be useful in Korea.     

Genetic Analysis of Avirulence/Virulence of an Isolate of Magnaporthe grisea from a Rice Field in Texas

ABSTRACT An isolate of Magnaporthe grisea, Tm4, from a rice field in Texas was crossed with a fertile laboratory strain, 70-6. The progenies showed segregation of avirulence/virulence on rice cvs. Newbonnet, Lemont, Lebonnet, Leah, and Katy. The avirulent/virulent segregation ratios were 29:6 on Newbonnet, Lemont, and Lebonnet; 28:7 on Leah; and 33:2 on Katy. There was cosegregation on the first three cultivars. Several avirulent progenies were backcrossed to virulent parent 70-6. Three generations of backcrossing avirulent progenies to 70-6 led to segregation ratios that suggested certain strains had only one avirulence gene. Strains avirulent only on cv. Katy or only on cvs. Newbonnet, Lemont, and Lebonnet were test crossed with virulent siblings. Strains that gave progeny ratios approximating 1 avirulent:1 virulent when crossed with virulent siblings were selected for further test crossing. Intercrosses between strains with possible single avirulence genes were made to determine whether these strains had the same or different avirulence genes. Many lines still segregated two genes for avirulence after three generations of backcrossing. This is based on the recovery of virulent progenies from crossing two avirulent siblings.     

Bacterial blight of rice and its control in Nepal

Research on Xanthomonas oryzae pv. oryzae, the bacterial blight of rice pathogen, was initiated at the Institute of Agriculture and Animal Science (IAAS) with the main objective of assessing the population structure of X. o. pv. oryzae through the use of both conventional and molecular markers in combination with virulence typing. A high DNA polymorphism was detected in the pathogen populations using different DNA probes and rep-PCR primers. Most strains were avirulent to cultivars containing the bacterial blight resistance gene Xa-21, which suggested the strategy that targets gene deployment is feasible in Nepal.     

Isoenzyme diversity in Xanthomonas campestris pv. mangiferaeindicae

A collection of 26 strains of Xanthomonas campestris pv. mangiferaeindicae isolated from three different host species in eight countries was investigated for variation in isozyme patterns. Three enzyme systems were analysed: esterase (EST), phosphoglucomutase (PGM) and superoxide dismutase (SOD). Four groups of strains were identified: nonpigmented strains isolated from mango and pepper-tree in Australia, Comores, India, Reunion Island, South Africa, and Taiwan; nonpigmented Brazilian strains from mango; nonpigmented strains from ambarella isolated in the French West Indies; heterogeneous yellow pigmented strains from mango (Brazil and Reunion Island). The value of isozyme profiling as markers of the pathogenicity groups in X. c . pv. mangiferaeindicae is discussed.     

The relationship between virulence and cytokinin production by Rhodococcus fascians (Tilford 1936) Goodfellow 1984

Qualitative and quantitative analyses were made of cytokinins secreted into liquid culture by virulent and avirulent strains of Rhodococcus fascians , and of the endogenous cytokinins in pea plants inoculated with the same strains. Both virulent and avirulent strains secreted a range of cytokinins into liquid culture. The non-hydroxylated cytokinins were present in the liquid cultures at levels up to 1000-fold higher than hydroxylated cytokinins, but the virulent strains secreted only slightly higher levels of isopentenyladenine (iP), isopentenyladenosine (iPA) and the tentatively identified methylthio-isopentenyladenosine (ms-iPA), than the avirulent strains. Pisum sativum cv. Novella plants that were inoculated with virulent strains contained only slightly higher levels of iP and ms-iPA but had lower levels of the cytokinin nucleotides than those plants that had been inoculated with the avirulent strains. The origin of the cytokinins secreted by avirulent and virulent strains is discussed, as is the significance of the cytokinin content of fasciated plants.     

Genetic Diversity of Xanthomonas oryzae pv. oryzae Strains from Sri Lanka

ABSTRACT Sixty strains of Xanthomonas oryzae pv. oryzae, collected from 29 locations in Sri Lanka in 1995, were analyzed by restriction fragment length polymorphism using either polymerase chain reaction-amplified 16S and 23S rDNA or the repetitive DNA element IS1112 from X. oryzae pv. oryzae as hybridization probes. Two different ribogroups were observed in the Sri Lankan strains using rDNA probes, whereas five clusters were identified by the IS1112 probe. Bootstrap analysis revealed that the five clusters defined by IS1112 were relatively robust. Our results suggest that the Sri Lankan strains are phylogenetically composed of five different groups. Each cluster was partially associated with climatic conditions (intermediate zone and wet zone) and was related to groups based on ribotyping. Based on virulence analysis using 12 rice cultivars, each containing a single resistance gene, 14 pathotypes were identified among the Sri Lankan strains. All strains were virulent to resistance genes Xa1, Xa2, Xa4, Xa10, Xa11, and Xa14. Only one strain (pathotype 1) was virulent to all major resistance genes including Xa21, while strains of the other pathotypes were all avirulent to Xa21. A partial relationship was found between the determined phylogenetic groups using the IS1112 probe and pathotypes for all but two clusters. The results of this study will facilitate the further understanding of the population structure of X. oryzae pv. oryzae in Sri Lanka.     

Isozyme and RAPD variation among Phytophthora colocasiae isolates from South-east Asia and the Pacific

Isozyme variation was studied in 94 isolates of Phytophthora colocasiae originating from Indonesia, Papua New Guinea, the Philippines, Thailand and Vietnam. Eight polymorphic enzyme systems (HK, PGM, PGI, Gluco, MDH, ICD, 6PDH, ME) revealed 52 isozyme patterns (zymotypes), each uniquely characterized by the presence or absence of 60 electromorphs. A core sample of 20 isolates was subsequently analysed with RAPD markers. Seven primers were used successfully and all profiles were reproducible. Clear bands were revealed and, in some cases, allowed differentiation between isolates exhibiting identical zymotypes. Results indicate that throughout this vast geographic region, taro leaf blight is caused by numerous and distinct strains that are genetically variable. Variation occurs within and between countries. The geographical distribution of zymotypes shows that none is common to two different countries. Although the differences in pathogenicity are not yet established, different P. colocasiae genotypes are likely to recombine and evolve rapidly as this species is heterothallic. From these results, a long-term breeding strategy is recommended for taro ( Colocasia esculenta ) based on recurrent selection using a wide genetic base composed of carefully selected parents from diverse geographic origins to maximize multigenic resistance in progenies.     

Antagonistic Interactions Between Strains of Xanthomonas oryzae pv. oryzae

ABSTRACT The ability of some phytopathogenic bacterial strains to inhibit the growth of others in mixed infections has been well documented. Here we report that such antagonistic interactions occur between several wild-type strains of the rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae. In mixed inoculations, a wild-type Philippine strain was found to inhibit the growth of a wild-type Korean strain. Furthermore, a nonpathogenic mutant of the Philippine strain maintained these antagonistic properties. Growth curve analysis indicated that both the wild-type Philippine strain and its nonpathogenic mutant inhibited the growth of the Korean strain 2 days after infection and prior to the onset of disease symptoms. When mixed with the nonpathogenic mutant, 10 out of 18 diverse wild-type X. oryzae pv. oryzae strains did not cause disease. Conversely, three of the strains that were not affected by the nonpathogenic mutant were found to inhibit the growth of both the wild-type and mutant Philippine strains, indicating that antagonism is widespread and strain specific. The observed growth inhibition occurred only in planta and did not correlate with bacteriocin activity in vitro. Antagonistic interactions also were found to affect resistance (R) gene-mediated resistance. The R gene Xa21 was capable of protecting rice plants coinoculated with nonantagonistic virulent and avirulent strains; however, when avirulent strains were coinoculated with virulent antagonistic strains, disease ensued. Taken together, these results indicate that X. oryzae pv. oryzae has evolved strategies to compete with rival strains in a fashion that allows virulent strains to evade R gene-mediated protection even when avirulent strains are present in the inoculum.     

青枯病原细菌无毒菌株诱导番茄防御酶系及粗提液抑菌活性的影响

 用紫外诱变法获得的青枯无毒菌株诱导番茄,研究了诱导番茄细胞内防御酶系及组织粗提液抑菌活性的影响。结果表明:番茄经无毒菌株处理后,体内与抗病反应相关的苯丙氨酸解氨酶(PAL)、过氧化物酶(POD)、多酚氧化酶(PPO)及超氧化物歧化酶(SOD)等防御酶系活性均显著增加。无毒菌株可诱导产生植株体外和体内对致病菌有抑制作用的抑菌物质。表明青枯无毒菌株产生诱导抗性的机制可能是激活了植物本身的抗病代谢过程。     

Changes in phenylalanine ammonia-lyase and peroxidase activities in wheat cultivars expressing resistance to the leaf-rust fungus

Phenylalanine ammonia-lyase (PAL) and peroxidase activities increased slightly during pre-haustorial stages of development of the leaf-rust fungus ( Puccinia recondita f. sp. tritici ) in each combination of rust strain and wheat line used. Activity of both enzymes greatly increased during the formation of the first haustoria by avirulent and virulent strains in an Lr28-bearing line but not in an Lr20 -bearing line. PAL increased markedly in activity at the time when an avirulent strain was known to cause hypersensitivity in the Lr28 -bearing line and when another avirulent strain was known to cause cellular changes that preceded the expression of resistance in the Lr20 -bearing line. PAL increased less markedly and not at all at corresponding times when virulent strains developed in the Lr28 and Lr20 lines, respectively. Increases in peroxidase activity occurred after the increases in PAL activity and were greater during resistance expression than in leaves infected with virulent strains. Cytochemical tests revealed enhancements of peroxidase activity in epidermal and guard cells in response to avirulent and virulent strains and in mesophyll cells that were becoming necrotic in response to avirulent strains only. The implications of the changes are that aromatic and phenylpropanoid synthesis and peroxidations may be enhanced in wheat by infection and particularly during resistance expression. The products of these processes may play roles in both Lr28- and Lr20 -based expression of resistance.     

Amoebiasis in foreign students

A total of 2,883 foreign students at the age of 18-30 years were examined for amoebiasis after their arrival to Czechoslovakia. Stool examinations revealed the presence of Entamoeba histolytica in 112 of them (3.9%). Students from 38 countries were found to be infected with this parasite. In a set of 2,064 students from these countries E. histolytica prevalence in stool was 5.4%. There were greater differences in the prevalence between individual countries inside a geographical region than between individual geographical regions. The highest E. histolytica prevalence in stool was found in students from tropical and southern Africa (6.7% of 745 examined) and the lowest in students from South-eastern Asia (3.1% of 321 examined). In a simple cross-section study, antibodies against E. histolytica were detected by enzyme-linked immunosorbent assay (ELISA) in the sera of 1,001 persons. Antibodies were detected in 7.9% of students at the following titres: 1:200 in 4.5%, 1:600 in 1.5%, 1:1,800 in 1.9%. Antibodies occurred more frequently in students carrying E. histolytica cysts (X2 = 14.9). Titre of ELISA antibodies in patients with confirmed liver abscess was higher than 1:1,800. counterimmunoelectrophoresis (CIEP) test was used for serum examinations of patients who had been demonstrated by ELISA to be seropositive and of those carrying E. histolytica cysts. In a set of 170 patients CIEP antibodies were also more frequent in those carrying E. histolytica cysts (X2 = 26.95). A comparison of the results of ELISA and CIEP tests in the same patients revealed that CIEP antibodies were more dependent on the actual parasitization with E. histolytica than ELISA antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)     

苏云金芽胞杆菌与绿僵菌对椰心叶甲的协同控制作用

测定了3个苏云金芽胞杆菌（Bacillus thuringiensis,Bt）菌株F6、NBT-18、Rt184与绿僵菌（Metarhizium anisopliae）菌株HK4复配剂对椰心叶甲致死作用。结果表明,3株Bt菌株对绿僵菌控制椰心叶甲的效果都有不同程度增强。在HK4孢子含量为106个/mL时,F6与HK4、NBT-18与HK4的复配剂处理椰心叶甲,5 d后死亡率分别达71.67%、61.35%,与单独用HK4分生孢子处理（死亡率为30.71%）相比,具有显著差异;而Rt184和HK4复配剂处理的死亡率与HK4单独处理无显著差异。在HK4孢子含量为107个/mL时,3种复配剂处理椰心叶甲5 d后,死亡率分别为82.74%、83.07%、68.61%,均与绿僵菌HK4单独处理（死亡率61.49%）有显著差异;且3个Bt菌株均能使绿僵菌HK4菌株侵染椰心叶甲的致死时间提前。     

Detection and analysis of inter- and intraspecific diversity of Pratylenchus spp. using isozyme markers

The root-lesion nematodes of the genus Pratylenchus are an economically important group of plant parasitic nematodes that show high similarity among sibling species. Isozyme patterns obtained by isoelectrofocusing (IEF) were used to differentiate and establish genetic relatedness among Pratylenchus species. A total of 40 populations comprising 9 Pratylenchus species and Radopholus similis from broad host and geographic origins was examined to compare isozyme patterns of esterase (EST), hexoquinase (HK), isocitrate dehydrogenase (IDH), malate dehydrogenase (MDH), phosphoglucose isomerase (PGI), phosphoglucomutase (PGM) and superoxide dismutase (SOD). Of these systems, only EST, MDH, PGI and PGM were useful for differentiation of P. vulnus , P. goodeyi , P. penetrans , P. scribneri , P. thornei and R. similis populations. The greatest intraspecific diversity was found within P. coffeae based on the isozyme patterns for MDH, PGI and PGM. Intraspecific variability was also detected among R. similis populations, which showed two isozyme patterns in EST and PGI systems. Less intraspecific variation was found within the P. penetrans group. The P. goodeyi population from Cameroon differed from the other populations in this specific group in its MDH, PGI and PGM phenotypes. Highly similar banding patterns of EST, MDH and PGI activity were found among the P. scribneri populations and the one population of P. agilis . A cluster analysis of the 40 populations, generated from the four enzyme banding patterns, produced groupings that broadly matched the previous classification into specific groups, reflecting intraspecific variability in some cases. The results confirm the potential use of isozyme patterns as markers for these nematode species and their value for diagnostic application.     

球孢白僵菌脂肪酸、酯酶、脂肪酶及其与毒力的关系

研究12株球孢白僵菌脂肪酸、酯酶和脂肪酶的产生水平及菌株对马尾松毛虫毒力间的关系。结果表明：酯酶产生水平（E）、脂肪酶产生水平（L）及豆蔻酸百分含量（X）等与白僵菌菌株对马尾松毛虫的毒力（LT50）间存在一定的相关性，其相关方程分别为：LT50=2．0931/（lgE-5．1165）＋0．4091；LT50=-6．68L^2＋31．9886L；LT50=13．5192-8．7042X。     

Differences in Virulence and Genomic Features of Strains of 'Candidatus Phytoplasma mali', the Apple Proliferation Agent

ABSTRACT Root and shoot samples from 24 symptomatic or nonsymptomatic apple trees infected with 'Candidatus Phytoplasma mali' were collected at different locations in Germany and France and used to inoculate rootstock M11 top grafted with cv. Golden Delicious. Inoculated trees were monitored over a 12-year period for apple proliferation (AP) symptoms and categorized as not or slightly, moderately, or severely affected. Based on symptomatology, the phytoplasma strains were defined as being avirulent to mildly, moderately, or highly virulent. Determination of phytoplasma titers by quantitative polymerase chain reaction (PCR) with DNA from roots revealed similar phytoplasma concentrations in all virulence groups. Molecular characterization of the strains by differential PCR amplification with five sets of primers resulted in 13 profiles. Six strains that were maintained in periwinkle and tobacco were molecularly characterized in more detail. The genome sizes of these strains as determined by pulsed-field gel electrophoresis using yeast chromosomes as size references ranged between 640 and 680 kb. Cleavage of the chromosome with the rare cutting restriction enzymes ApaI, BamHI, BssHII, MluI, and SmaI resulted in macro fragment patterns distinctly different in all strains. Similar results were obtained by Southern blot hybridization with three probes derived from strain AT. Differential PCR amplification at an annealing temperature of 52 degrees C using eight primer pairs derived from strain AT revealed heterogeneity of target sequences among all strains. Based on these results, there is considerable variability in virulence and genomic traits in 'Ca. P. mali'. However, correlations between molecular markers and virulence or phytoplasma titer could not be identified.     

Glomerella cingulata on camellia

Glomerella cingulata was shown to be associated with a leaf blotch, canker and dieback of imported camellia plants. It was particularly damaging on hybrids of Camellia saluenensis such as C. x williamsii cv. Donation. Pathogenicity tests, using whole plants and detached leaves of cv. Donation, distinguished between virulent and a virulent strains of the fungus. The virulent strain did not cause severe damage when inoculated into a range of other woody ornamentals. Conversely, Colletotrichum spp., including G. cingulata , isolated from other hosts were not virulent to Camellia cv. Donation. Apart from a loss of virulence associated with a mutation to paleness of colonies in culture, virulent isolates with dark-grey colony centres were morphologically identical to avirulent isolates from camellia, including tea plant ( Camellia sinensis ), and to some isolates from other hosts. A form of Colletotrichum acutatum isolated from camellia and readily distinguished from G. cingulata by bright pink colonies and conidial shape was shown to be avirulent on Camellia cv. Donation. It is proposed that the virulent form on ornamental camellia be described as Glomerella cingulata f.sp. camelliae.