Comparative proteomic profiles of Pinus monticola needles during early compatible and incompatible interactions with Cronartium ribicola

The proteomic profiles of primary needles from Cr2-resistant and cr2-susceptible Pinus monticola seedlings were analysed post Cronartium ribicola inoculation by 2-DE. One hundred-and-five protein spots exhibiting significant differential expression were identified using LC–MS/MS. Functional classification showed that the most numerous proteins are involved in defence signalling, oxidative burst, metabolic pathways, and other physiological processes. Our results revealed that differential expression of proteins in response to C. ribicola inoculation was genotype- and infection-stage dependent. Responsive proteins in resistant seedlings with incompatible white pine blister rust (WPBR) interaction included such well-characterized proteins as heat shock proteins (HSPs), reactive oxygen species (ROS) scavenging enzymes, and intermediate factors functioning in the signal transduction pathways triggered by well-known plant R genes, as well as new candidates in plant defence like sugar epimerase, GTP-binding proteins, and chloroplastic ribonucleoproteins. Fewer proteins were regulated in susceptible seedlings; most of them were in common with resistant seedlings and related to photosynthesis among others. Quantitative RT-PCR analysis confirmed HSP- and ROS-related genes played an important role in host defence in response to C. ribicola infection. To the best of our knowledge, this is the first comparative proteomics study on WPBR interactions at the early stages of host defence, which provides a reference proteomic profile for other five-needle pines as well as resistance candidates for further understanding of host resistance in the WPBR pathosystem.     

Antifungal activity of a Pinus monticola antimicrobial peptide 1 (Pm-AMP1) and its accumulation in western white pine infected with Cronartium ribicola

Pinus monticola antimicrobial peptide 1 (Pm-AMP1) was expressed and purified from bacterial cell lysate and its identity and purity confirmed by Western blot analysis using the Pm-AMP1 antibody. Application of Pm-AMP1 resulted in visible hyphal growth inhibition of Cronartium ribicola , Phellinus sulphurascens , Ophiostoma montium , and Ophiostoma clavigerum 3-12 days post-treatment. Pm-AMP1 also inhibited spore germination of several other phytopathogenic fungi by 32%-84% 5?days post-treatment. Microscopic examination of C. ribicola hyphae in contact with Pm-AMP1 showed distinct morphological changes. Seven western white pine ( Pinus monticola Douglas ex D. Don) families (Nos. 1, 2, 5, 6, 7, 8, 10) showing partial resistance to C.?ribicola in the form of bark reaction (BR) were assessed by Western immunoblot for associations between Pm-AMP1 accumulation and family, phenotype, canker number, and virulence of C. ribicola. There was a significant difference (p?< 0.001) in mean Pm-AMP1 protein accumulation between families, with higher levels detected in the full-sib BR families (Nos. 1, 2, 5) than the half-sib BR families (Nos. 6, 7). Family 8, previously described as a Mechanism 'X' BR family, had the highest number of BR seedlings and displayed high Pm-AMP1 levels, whereas the susceptible family (No. 10) showed the lowest levels (p?< 0.05). Family 1 showed a significant association between Pm-AMP1 accumulation and overall seedling health (p?< 0.01, R?= 0.533), with higher protein levels observed in healthy versus severely infected seedlings. In general, low Pm-AMP1 levels were observed with an increase in the number of cankers per seedling (p?< 0.05), and seedlings inoculated with the avirulent source of C. ribicola showed significantly higher Pm-AMP1 levels (p?< 0.05) in the majority of BR families. Cis-acting regulatory elements, such as CCAAT binding factors, and an AG-motif binding protein were identified in the Pm-AMP1 promoter region. Multiple polymorphic sites were identified within the 5' untranslated region and promoter regions. Our results suggest that Pm-AMP1 is involved in the western white pine defense response to fungal infection, as observed by its antifungal activity on C. ribicola and a range of phytopathogens as well as through its association with different indicators of resistance to C.?ribicola.     

Genetic diversity in populations of Cronartium ribicola in plantations and natural stands of Pinus strobus

Genetic diversity was studied in 22 populations of the white pine blister rust fungus Cronartium ribicola from natural stands and plantations of eastern white pine, Pinus strobus. Pseudo-allelic frequencies were estimated at each of 7 putative RAPD loci by scoring for presence or absence of amplified fragments in dikaryotic aecidiospores. Analysis of genetic distance between all pairs of populations did not reveal any trend with regard to geographic origin or type of white pine stand. In addition, when hierarchical population structure was analysed, total genetic diversity (H _s =0.214) was mostly attributable to diversity within populations (H _s =0.199; AMOVA _st =0.121, P<0.01). Genetic diversity of populations relative to region of origin (east, centre, and west) or type of stand (natural stands vs plantations) was not significantly different from zero (P>0.10) Nevertheless, a significant proportion of genetic differentiation was found between populations within region or stand type (F _st =0.114; _sc =0.132, P<0.001). This result indicates that some population structure exists but that it appears to be independent of region of origin or type of stand. At least for 2 populations from white pine plantations, it appears possible that a recent introduction of a limited number of propagules was responsible for low levels of genetic diversity. We interpret these results as meaning that either long-distance dispersal is taking place between populations more than 1000 km apart or that these populations share a common recent ancestor. In addition, we suggest that C. ribicola may still be expanding its distribution by colonizing new plantations.     

Pinus monticola pathogenesis-related gene PmPR10-2 alleles as defense candidates for stem quantitative disease resistance against white pine blister rust (Cronartium ribicola)

White pine blister rust, caused by the invasive fungus Cronartium ribicola, has been responsible for extremely high mortality of native western white pine (Pinus monticola) and other five-needle pines in natural stands throughout western North America. The presence of this non-native fungus has also led to greatly restricted use of western white pine for reforestation. A few families of defense proteins have been found as functional candidates involved in tree resistance to rust infection. Here we report genetic variation of a gene encoding a family 10 pathogenesis-related (PR) protein (PmPR10-2) in open-pollinated seed families with different levels of stem quantitative disease resistance (QDR). Six novel alleles and five common genotypes were identified inside the PmPR10-2 locus: these genetic variations included 33 single nucleotide polymorphisms (SNPs) throughout the gene regions and copy variation of a rare octanucleotide simple sequence repeat (SSR), 5′-AATTATTT-3′, in the gene intron. PmPR10-2 exhibited a moderate level (average r ²?=?0.42) of linkage disequilibrium. Two-thirds of the identified SNPs and the SSR marker were significantly associated with stem QDR levels. The PmPR10-2 genotype (H3:H3) exhibited the highest level of stem QDR (P?<?0.01). Cost-effective and co-dominant SSR markers were developed and used for genotyping the PmPR10-2 locus using simple PCR, providing a potential molecular tool for accelerating screening efforts of white pine resistance against C. ribicola.     

Survival of Cronartium ribicola on a medium containing host tissue culture diffusates

Vegetative mycelia ofCronartium ribicola J. C. Fisch. exRabenh. that were removed from media surrounding infected pine host tissue cultures did not survive when transplanted to a defined artificial medium. Similar mycelia retained on a medium that had formerly supported pine host tissue cultures did survive for periods up to 120 days. These mycelia were fully capable of reinfecting healthy host tissue cultures.

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Zusammenfassung Vegetative Myzelien vonCronartium ribicola J. C. Fisch. exRabenh., die von Nährmedien, die die Gewebskultur des infizierten Kiefernwirtes umgeben, entfernt worden sind, überlebten nicht, wenn sie auf einen definierten, künstlichen Nährboden übertragen worden sind. Ähnliche Myzelien auf einem Nährboden gehalten, der vorher Kiefer-Gewebskultur getragen hat, überlebten für eine Periode bis zu 120 Tagen. Diese Myzelien waren fähig, gesunde Wirtsgewebekulturen wiederum zu infizieren.

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stationed in Moscow, Idaho, at Forestry Sciences Laboratory, maintained in cooperation with the University of Idaho.     

抗感枯萎病西瓜根际微生物比较研究

本文通过传统微生物培养方法,研究了在土培和基质培条件下,抗感枯萎病西瓜不同生育期根际和非根际微生物数量的变化.结果表明,微生物数量在根际显著高于在非根际微生物,且随西瓜生长发育的阶段不同而变化,苗期根际微生物数量最少,以后随西瓜生长发育,根际微生物数量不断增加,至生长旺盛的开花结果期,微生物数量达到最高,在西瓜生长发育后期,根际微生物数量又有所回落.西瓜抗枯萎病性与根际细菌的数量具有相关性,在生长发育各个阶段,无论是土培还是基质培,均表现为抗病材料的根际细菌数量高于感病材料的根际细菌数量.根际真菌与放线菌数量与西瓜的抗感枯萎病性没有相关性.非根际微生物数量在整个生育期变化辐度较小.非根际细菌数量在土培条件下几乎保持在同一水平,在基质培条件下迅速增加,至生长后期有所回落.非根际真菌与放线菌数量在土培和基质培条件下均于生长后期达到最高.     

The occurrence of urediniospores of Cronartium ribicola within the petiole of Ribes petiolare

Urediniospores ofCronartium ribicola J. C. Fisch. exRabenh. were observed within the petiole ofRibes petiolare L. These spores occurred internal to xylem tissues within the region of undifferentiated parenchyma cells (pith). Fungal tissues were sparse and fully differentiated sori were absent.Research Plant Pathologist and Biological Laboratory Technician, USDA Forest Serv., Intermountain Forest and Range Exp. Sta., Ogden, Utah, stationed in Moscow, Idaho83843, at Forestry Sciences Laboratory, maintained in cooperation with the University of Idaho.     

抗感枯萎病西瓜根际细菌群落多样性比较

本文通过传统微生物培养方法,结合现代分子生物学技术对抗感枯萎病西瓜根际可培养细菌群落进行了比较研究.结果表明,抗病西瓜根际可培养细菌的多样性要高于感病西瓜,且细菌分布的均匀度也高于感病西瓜.表现为抗病西瓜根际可培养细菌的多样性指数H'(1.29)和1/D(30.28)分别高于感病西瓜的H'(1.12)和1/D(2.482).抗病西瓜根际可培养细菌的均匀度指数E(0.72)也高于感病西瓜的E(0.69).抗感西瓜根际分别具有不同的可培养优势群落,抗病西瓜根际可培养优势基因型为基因型1,占51.1%,感病西瓜根际可培养优势基因型为基因型7,占58.7%.     

Destructive Effects of a Mycoparasite Trichoderma atroviride SS003 on Aeciospores of Cronartium ribicola

The white pine blister rust caused by Cronartium ribicola is one of the most severe diseases of Pinus armandii Franch in Yunnan Province, China, and controlling the disease is very difficult. A mycoparasite (Trichoderma atroviride P. Karsten SS003) we isolated can effectively destroy aeciospores. Microscopic analysis showed that aeciospore warts started to fall off 3 days after SS003 inoculation, and the outer wall of the aeciospores was deformed and completely broken 5 days after treatment. SS003 treatment indoors and in the field was effective against C. ribicola. SS003 mycelium grew well on aeciospore piles, and the outer walls of most aeciospores were broken when examined by microscope. The average efficacy of SS003 against Armandii pine blister rust reached 71.85% after continuous treatment for 1.5 years in the field. Additionally, safety tests showed that P. armandii seedlings experienced no side effects when they were inoculated with either conidial suspensions or mycelium solution of SS003. Our results suggest that T. atroviride SS003 is a promising mycoparasite for controlling Armandii pine blister rust.     

Electrophoretic Comparison of Mitochondrial Polypeptides from Maize Lines Susceptible and Resistant to Helminthosporium maydis Race T

The mitochondrial polypeptides from maize lines susceptible and resistant to Helminthosporium maydis were studied by single- and two-dimensional gel electrophoresis. Approximately 120 polypeptides were detected by two-dimensional gel electrophoresis. However, it was not possible to detect qualitative differences between the mitochondrial polypeptides of the two inbreds. These observations are discussed with reference to the putative mechanism of action of the pathotoxin.     

A Comparison of Soybean Agglutinin in Cultivars Resistant and Susceptible to Phytophthora megasperma var. sojae (Race 1)

The amount of soybean agglutinin (SBA) detectable by radioimmunoassay in seeds of resistant cultivars to Phytophthora megasperma var. sojae was approximately twice that of susceptible cultivars. SBA was preferentially released at earlier times (6-9 hours) and in higher amounts in the imbibate from resistant cultivars as compared to susceptible cultivars. The lectin in the imbibate was immunologically identical to the seed lectin, indicating little or no proteolysis had occurred, and was active in hemagglutination. Binding of fluorescein isothiocyanate-labeled SBA to mycelial cell walls could be abolished by adding N-acetyl galactosamine or galactose. Purified SBA at concentrations of 150 to 300 micrograms inhibited mycelial growth by 50%, and the imbibate from Govan (resistant) cultivar was more inhibitory than the imbibate from Shore (susceptible) cultivar. Removal of SBA from the imbibate by affinity chromatography abolished the inhibition of mycelial growth, but the inhibition could be recovered from the eluant containing lectin.     

Phenols in Cotton Seedlings Resistant and Susceptible to Alternaria macrospora

In healthy cotton seedlings, stems have a lower phenol content than leaves, but resistant plants have an altogether relatively higher phenol content than susceptible plants. Phenols extracted from infected plants can inhibit the growth of A. macrospora in vitro. In cotton plants infected with A. macrospora, phenols are oxidized by polyphenoloxidase rather than peroxidase and catalase. The main oxidative activity was around the developing necrotic area but activity was detected far from, necrosis as well. Though pre–inoculation mechanical injuries operated the phenol oxidation mechanism in the plant, they neither prevented nor encouraged the increase in disease severity. Isozyme pattern showed that contribution of all participants in the pathological interaction to the oxidative mechanism occurred in the diseased plant. A negative linear correlation was found between polyphenoloxidase activity, phenol accumulation and resistance. This study suggests that the phenol oxidative mechanism, participates in cotton plant resistance to A. macrospora.     

Cloning and characterization of a cDNA of cro rI from the white pine blister rust fungus Cronartium ribicola

White pine blister rust (WPBR) is caused by the fungus Cronartium ribicola which has five spore stages on two unrelated hosts, the five-needle pines and Ribes spp. Recently, during the molecular analysis of the proteins and genes involved in host-pathogen interaction, the WPBR fungal protein Cro rI was identified in infected white pine tissues. To further characterize Cro rI, an expression cDNA library from poly(A)(+) mRNA of C. ribicola axenic mycelial culture was constructed and immunoscreened and the cDNA was cloned. Sequence analysis indicated an open reading frame of 462 bases, which encodes a protein of 153 amino acid residues with a molecular mass of 16.7 kDa and a predicted isoelectric point (pI) of 8.93. Based on the N-terminal amino acid sequences of Cro rI, the secreted portion of Cro rI protein should be 136 amino acids long with several putative posttranslational modification sites and a molecular mass of 14.8 kDa. The predicted pI for the secreted portion was 9.34. The predicted N-terminal signal peptide was 17 amino acids long. The N-terminal 42-amino acid sequence of the predicted mature protein (secreted portion) was identical to the amino terminal sequence of Cro rI that was previously determined. Southern blot hybridizations indicated that the C. ribicola genome contained at least two copies of the cro rI gene. Isolation of the genomic PCR fragment, which was approximately 400 bp longer than the cDNA, and subsequent cloning and sequencing analyses confirmed that there were three introns within the coding regions. Western immunoblot analyses revealed that Cro rI protein accumulated in large amounts only in the infected white pine tissues while no trace was detectable in the alternate Ribes stage or the five different spores, suggesting a critical role of Cro rI in the haploid stage of the fungus (in pine). The translocation of Cro rI was only found to occur in cankered trees, and not in the young infected seedlings. The implications of Cro rI in pathogenesis are discussed.     

裂果易发性不同的荔枝品种果皮中细胞壁代谢酶活性的比较

“糯米糍”荔枝裂果率极显著高于“淮枝”,前者果皮中的果胶酶、纤维素酶和果胶甲酯酶的活性高于后者,其中以果胶酶活性差异最明显,其次是纤维素酶,果胶甲酯酶差异最小;“糯米糍”细胞壁结合型的过氧化物酶(POD)和多酚氧化酶(PP0)活性明显高于“淮枝”,而水溶性POD和PP0的活性则两个品种间无明显差异。据此认为,果皮细胞壁水解酶活性以及细胞壁结合型的POD和PPO的活性高的荔枝品种,其裂果率也高。文章对细胞壁代谢相关酶类在果皮抗裂性形成中的作用进行了讨论。