Anatomy of cranberry stem gall and localization of bacteria in galls

ABSTRACT Cranberry stem gall is characterized by tumors that girdle stems, thereby killing all distal leaves, flowers, and fruit. Bacteria that produce high levels of the plant growth hormone indole-3-acetic acid (IAA) are associated with and believed to cause cranberry stem gall. The anatomy of naturally occurring galls on woody cranberry plants and galls caused by inoculation of micropropagated cranberry plants with Pantoea agglomerans strain 4/99 was consistent with elevated levels of IAA in plants. Field galls exhibited hypertrophy and hyperplasia of tissue external to the vascular cambium, resulting in extensive stem swelling and splitting of the periderm. Similarly, galls on micropropagated plants contained enlarged cortical parenchyma cells. The current year's xylem vessels in field galls were narrow and dense compared with xylem vessels of healthy stems. Curved xylem elements apparently developed de novo within field galls and galls on inoculated plants. Cavities and fissures in both types of galls contained dense aggregates of bacteria. Treatment of micropropagated plants with synthetic IAA caused hypertrophy of cortical parenchyma and formation of adventitious roots. The results support the hypothesis that IAA-producing bacteria cause cranberry stem gall.     

Bacterial populations associated with rice seed in the tropical environment

ABSTRACT During the 1995 wet season, harvested rice seed was collected from farmers' fields at different locations in Iloilo, Philippines. Bacterial isolations from crushed seed yielded 428 isolates. The isolates were characterized by BOX-polymerase chain reaction fingerprinting of total genomic DNA and represented 151 fingerprint types (FPT). Most FPTs were found on a single occasion, although matching fingerprints for isolates from different samples also were found. Identifications were made by cellular fatty acid methyl ester analysis and additional use of Biolog GN/GP MicroPlates and API 20E/50CHE systems. The predominant bacteria were Enterobacteriaceae (25%), Bacillus spp. (22%), and Pseu-domonas spp. (14%). Other bacteria regularly present were identified as Xanthomonas spp., Cellulomonas flavigena, and Clavibacter michiganense. Of the total number of isolated bacteria, 4% exhibited in vitro antifungal activity against Rhizoctonia solani or Pyricularia grisea. Two percent of isolates were pathogens identified as Burkholderia glumae and Burkholderia gladioli. Five percent of isolates induced sheath necrosis on only 50 to 90% of inoculated plants and were related to Bacillus pumilus, Paenibacillus spp., Pseudomonas spp., and Pantoea spp.     

番茄青枯病内生拮抗细菌的筛选

 从广西一些市县采集番茄茎标本分离得到55个细菌菌株,分属为芽孢杆菌(Bacillus spp.)、黄单胞菌(Xanthomonas spp.)、假单胞菌(Pseudomonas spp.)和欧文氏菌(Erwinia spp.),其中芽孢杆菌为优势种群。经回接测试,有36个菌株为番茄植株内生菌。这些内生菌只有7个菌株对番茄青枯病菌有拮抗作用,芽孢杆菌B₄₇菌株对番茄青枯病菌拮抗作用较强,经室内和田间初步防治测定,它对番茄青枯病有较好的防治效果。     

葡萄根癌土壤杆菌的不同生化型对葡萄及其它植物致病性的初步研究

 从北京、内蒙古、吉林、辽宁、山东等地采到的葡萄根癌病标本中,分离到根癌土壤杆菌[Agrobacterium fumefaciens(Smith&Town.) Conn]3种生化型菌株。它们对玫瑰香葡萄的致病性明显不同。生化Ⅲ型菌株致病性强,形成大、中型瘤。生化Ⅰ型、Ⅱ型菌株致病性弱,只形成小瘤。生化Ⅲ型的不同菌株对葡萄、向日葵、烟草、番茄等8种植物的致病性也很不同。一类只能侵染葡萄,是窄寄主型;另一类除葡萄外,还能侵染向日葵、烟草、番茄,是宽寄主型。玫瑰香、龙眼等葡萄对3种生化型菌株比较敏感;莎巴珍珠、贝达等比较抗病。生化Ⅲ型和Ⅰ型的一些菌株也能侵染梨、李、杏、樱桃、榆叶梅等几种果树。从山东白雅葡萄上分离的BS33-10菌株(胭脂碱质粒类型)接种在烟草、番茄上能形成畸嵌瘤。     

Crown gall of tobacco caused by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> biovar 1 in tobacco fields

Crown gall disease of tobacco was found in Iwate Prefecture, Japan in 1995. Ten bacterial isolates, obtained from the galls of tobacco, were identified as Agrobacterium tumefaciens (Smith and Townsend 1907) Conn 1942 biovar 1 based on their ability to induce galls on the 14 tested plants, including tobacco after needle-prick inoculation, and on 12 cultural, physiological, and biological characteristics. The growth of the causal organism was not inhibited in vitro by agrocin of A. radiobacter strain K84. This report is the first on the natural occurrence of crown gall caused by A. tumefaciens on tobacco plants.     

月季根癌病病原菌分离及抗病资源初步筛选

从月季品种金玛丽、曼海姆、杏花村、梅郎口红的根部肿瘤组织中纯化了9株分离物,根据其在MW选择性培养基上的单菌落形态初步判断其为根癌土壤杆菌。以根癌土壤杆菌高度保守的virD2和ipt基因的部分序列设计引物对分离的菌株进行PCR检测,其中有6株菌株能扩增出virD2和ipt基因片段,为根癌土壤杆菌毒性菌株。采用针刺涂抹法接种向日葵、荷花蔷薇幼茎,6株菌株均能在供试植株上形成肿瘤,一个月后肿瘤大小有显著差异,说明分离获得的根癌土壤杆菌株毒性有差异。以强毒性菌株J-5-1在田间接种了部分蔷薇属野生资源,以鉴定其对根癌病的抗性。根据发病率、肿瘤大小、肿瘤干质量以及感病后植株生长状况将植物的根癌病抗性分为高度抗病、中度抗病、中度感病、高度感病4个类型。     

Occurrence of indole-3-acetic Acid-producing bacteria on pear trees and their association with fruit russet

ABSTRACT A relatively high percentage of epiphytic bacteria on pear leaf and fruit surfaces had the ability to produce indole-3-acetic acid (IAA) in culture media supplemented with tryptophan. While over 50% of the strains produced at least small amounts of IAA in culture, about 25% of the strains exhibited high IAA production as evidenced by both colorimetric and high-performance liquid chromatography analysis of culture supernatants. A majority of the strains that produced high amounts of IAA were identified as Erwinia herbicola (Pantoea agglomerans), while some strains of Pseudomonas syringae, Pseudomonas viridiflava, Pseudomonas fluorescens, Pseudomonas putida, and Rahnella aquaticus that produced high amounts of IAA also were found on pear. Fruit russeting was significantly increased in 39 out of 46 trials over an 8-year period in which IAA-producing bacteria were applied to trees compared with control trees. A linear relationship was observed between fruit russet severity and the logarithm of the population size of different IAA-producing bacteria on trees in the 30 days after inoculation, when normalized for the amount of IAA produced by each strain in culture. On average, the severity of fruit russet was only about 77% that on control trees when trees were treated at the time of bloom with Pseudomonas fluorescens strain A506, which does not produce IAA. Both total bacterial populations on pear in the 30-day period following full bloom and fruit russet severity varied greatly from year to year and in different commercial orchards over a 10-year period. There was a strong linear correlation between the logarithm of total bacterial population sizes and fruit russet severity.     

土壤杆菌K1026对果树根癌病的生物防治

由致病性根癌土壤杆菌Agrobacterium spp.侵染引起的根癌病对山东及我国其他省份的樱桃、桃及其他果树生产造成严重的影响.土壤杆菌Agrobacterium rhizogenes K1026在澳大利亚已制成生防菌剂并在全球多个国家实现商品化,但在我国果树育苗和生产行业中还未得到广泛应用.本文从山东樱桃和桃树根癌组织及感病土壤中分别分离到100余株致病性土壤杆菌,经鉴定均属于生物Ⅱ型土壤杆菌;利用双层平板抑菌试验,检测了37株病原菌对菌株K1026的敏感性,结果显示,所有菌株均对K1026产生的抗生物质敏感,可以在Stonier,s培养基上产生5种以上不同类型的抑菌圈;胡萝卜切片试验显示,菌株K1026可完全抑制37株病原菌对胡萝卜的致瘤活性.上述结果为土壤杆菌K1026用于山东省樱桃及桃树根癌病的生物防治奠定了基础.     

Anastomosis group and pathogenicity of isolates of Rhizoctonia solani from potato crops in South Australia

Isolates of Rhizoctonia collected from the stems, roots, tuber sclerotia and soil of potato crops in Virginia and Lenswood, South Australia, were identified to anastomosis groups (AG). Of the 301 multinucleate isolates of Rhizoctonia solani tested, 90% were AG-3, 7% were AG-4 and 2% were AG-5; 12 isolates were binucleate Rhizoctonia spp. This is the first report of isolates of AG-4 and AG-5 causing disease in potato crops in South Australia. All AG-3, AG-4 and AG-5 isolates tested caused rhizoctonia disease symptoms on the potato cultivar Coliban in pathogenicity trials conducted under glasshotise conditions. Both AG-3 and AG-5 isolates caused black scurf and stem cankers, although symptoms of black scurf were less severe with AG-5. AG-4 isolates produced the most severe stem and stolon cankers of all isolates tested. The pathogenicity of tuber-borne inoculum was confirmed by growing plants from sclerotia-infested tubers. AG-8 isolates from diseased barley and wheat produced severe root cankers and caused loss of feeder roots on inoculated potato plants. Results suggest that rhizoctonia disease in potato fields in South Australia is caused by a combination of different anastomosis groups and this has important implications for crop rotations.     

云木香内生细菌的分离鉴定及其对大丽轮枝菌的抑制作用

从药用植物云木香中分离得到41株内生细菌, 采用细菌16S rRNA基因测序的方法对这些菌株进行鉴定, 结果显示, 这些云木香内生细菌分别属于6个属, 其中假单胞菌属Pseudomonas和拉恩氏菌属Rahnella为优势菌属。采用对峙培养方法对这些菌株进行筛选, 发现其中12株内生细菌对大丽轮枝菌Verticillium dahliae具有抑制作用, 其中有2株属于拉恩氏菌属Rahnella, 7株属于假单胞菌属Pseudomonas, 3株属于沙雷氏菌属Serratia。12株内生细菌对棉花黄萎病的温室防效评估结果表明, 假单胞菌属菌株R1-6、R1-13和S1-2对棉花黄萎病的温室防效达到55%以上, 具有潜在的生产利用价值。     

Identification of a bacterium isolated from galls on carrot and weeds

In 2004, bacterial galls were found on the roots of carrots in Shizuoka Prefecture, Japan. Galls were about 0.1–2 cm in diameter, light brown in color and had rough surfaces. In 2005, similar galls were found on the roots of three weeds: henbit (Lamium amplexicaule L.), Persian speedwell (Veronica persica Poir.) and leaf mustard (Brassica juncea L.). A bacterium that forms white, rough colonies was isolated from the carrot and weeds galls. The bacterial isolates had properties identical with Rhizobacter dauci Goto and Kuwata. Phylogenetic analysis based on 16S rDNA sequences showed that the carrot isolate had the highest homology (similarity of 100%) with that of the type strain of R. dauci. Rep-PCR genomic fingerprinting using BOX A1R primer showed that the carrot and weeds isolates were nearly identical. Pathogenicity of the isolates was confirmed by inoculating the roots of carrots and the weeds. After 2–5 weeks, they formed galls on the roots of the original host species and on other plant species tested. The galls were indistinguishable from those formed naturally, and the inoculated bacterium was reisolated. Thus, the causal bacterium of carrot and weeds gall was identified as R. dauci, and the bacterium was found to have a wider host range than previously known. These weed hosts may serve as inoculum sources for carrot bacterial gall disease.     

Identification and pathogenicity of Fusarium species associated with pokkah boeng of sugarcane in Brazil

Brazil is the world’s biggest producer of sugarcane (Saccharum spp. hybrids). Pokkah boeng is an important fungal disease in this crop caused by members of the Fusarium fujikuroi species complex (FFSC) and characterized by deformation of the aerial part of the plant and stem rot. While the occurrence of symptoms has been reported in plantations in Brazil, no official reports of the disease exist. In this study, species of the FFSC were identified that are associated with sugarcane plants with symptoms of pokkah boeng in Brazil. This was achieved using two-loci molecular phylogeny, sexual compatibility and analysis of morphological markers. The ability of strains to cause disease in plants of sugarcane, maize, sorghum and millet was also evaluated. The 39 isolates studied were identified as F. sacchari, F. proliferatum and another, still unknown, phylogenetic lineage that is sister to F. andiyazi. Crossing field isolates of F. sacchari and F. proliferatum with their respective tester strains produced fertile perithecia and viable ascospores. All three species induced symptoms of pokkah boeng on inoculated sugarcane plants and caused stem rot in maize, sorghum and millet. Symptoms on sugarcane are chlorosis and necrosis of leaves, punctured lesions, twisted leaves, reduction of the total leaf area, death of the top of the plant and stalk rot. The findings confirmed the aetiology of the disease in Brazil, generated basic knowledge for the development of strategies for diagnosis and monitoring of the disease and support breeding programmes for selecting resistant germplasm.     

进境澳大利亚豌豆细菌性萎蔫病病原菌的分离鉴定

 2008年3月自澳大利亚进口的豌豆种苗病变部位分离到致病性菌株,该菌株能引起豌豆茎部、卷须萎蔫腐烂,叶片褪绿等症状。针刺接种豌豆,发生的病变状态与自然发病的症状相同,且在接种发病植株中又分离到形态上一致的病原菌,得到了柯赫法则的验证。经形态特征、培养性状、生理生化及16S rDNA序列分析,鉴定其为桃色欧文氏菌(Erwinia persicina)。这是我国首次从进境豌豆中检出该病害。     

Evidence of Migration and Endophytic Presence of Agrobacterium tumefaciens in Rose Plants

Agrobacterium tumefaciens was isolated from stem tumors of several rose cultivars showing that the bacterium is the causal agent of aerial galls in rose plants. No differences were observed in the characteristics of the Agrobacterium isolates from crown or aerial galls. Stem inoculation of ten rose cultivars showed that all of them were susceptible to A. tumefaciens but differences in the size of the resulting tumors were observed. The movement of A. tumefaciens in rose plants was demonstrated using two wild type strains and two antibiotic resistant mutants. Three months after inoculation, the inoculated strains were recovered in the roots, crown and below and above the inoculation site but low numbers of pathogenic Agrobacterium cells were isolated. New tumors appeared in 5% of the noninoculated wounds. A. tumefaciens was isolated from the stem at different distances from the tumor in naturally infected plants. In symptomless commercial plants, the isolation from the roots, crown and at different stem levels demonstrated the existence of systemic and latent infections in rose. Direct isolation using a nonselective and selective media with or without a previous enrichment step were efficient methods for isolating tumorigenic Agrobacterium from the different parts of rose plants.     

Interaction of Root-knot Nematodes (RKN) and the Bacterium Agrobacterium Tumefaciens in Roots of Prunus Cerasifera: Evidence of the Protective Effect of the Ma RKN Resistance Genes Against Expression of Crown Gall Symptoms

Agrobacterium tumefaciens (AT) is the causal agent of crown gall, a major problem in the family Rosaceae and particularly for Prunus spp. Crown gall symptoms result from the bacterial infection of the cells damaged mechanically at the collar or by root parasitic nematodes. Myrobalan plum (P. cerasifera) is susceptible to AT and is not a host for the root-knot nematode (RKN), M. hapla. Some clones of this plum carry single Ma resistance genes that control M. arenaria, M. incognita and M. javanica. The four above mentioned RKN and Myrobalan progenies segregating for Ma were used in experiments aimed at obtaining a better knowledge of the interaction between AT and RKN in relation to the RKN resistance genes. Prunus rooted cuttings, naturally infected with the bacterium were repotted, grown and inoculated individually with RKN. In a first experiment, Prunus plants were (i) either inoculated with 10,000 juveniles (J2s) of M. arenaria to provide a short inoculum pressure (SIP) or (ii) inoculated by association with one M. arenaria-galled tomato root system that produced a high and durable inoculum pressure of the same nematode species. Four months after RKN inoculation, plants were rated for nematode and bacterial root galling symptoms. RKN and AT galls were more numerous and more homogenous under DIP than under SIP. Nevertheless, for both inoculum regimes, AT galls were present in the RKN-susceptible clones (= carrying none of the Ma genes) and absent in the RKN-resistant clones. Subsequent experiments, conducted under DIP with M. arenaria, M. incognita, M. javanica and M. hapla, also showed, for the three first species, the presence of AT galls only in RKN-susceptible clones whereas Prunus plants inoculated with M. hapla and nematode-free controls were free of AT galls. Consequently RKN act as a wound agent in the AT infection process of Myrobalan plum only when the plant develops a compatible reaction (i.e. when it lacks the Ma resistance genes). Considering that J2s do penetrate the roots of resistant plants, the absence of crown gall symptoms on this material even under durable inoculum pressure strengthens the hypothesis that this nematode stage has a very weak effect on plant cells during the infection process. This is the first evidence of the protective effect of a RKN resistance gene against the expression of root crown gall consecutive to RKN infection. The protective effect of Ma and presumably of other RKN resistance genes against AT is a strong argument for their introgression into Prunus and other Rosaceae or bacterium-susceptible crops.     

Histological analysis of spindle and spheroid root galls caused by Plasmodiophora brassicae

Clubroot of crucifers, caused by the obligate parasite Plasmodiophora brassicae, is characterized by the formation of conspicuous root galls. These galls usually have a club- or spindle-shaped morphology, and interfere with water and nutrient uptake by infected plants. Smaller galls, historically regarded as resistance structures and distinct from the typical spindle-shaped galls, have also been identified and termed ‘spheroid galls’ because of their spherical or nearly spherical form. An assessment of various Brassica species and varieties revealed that spheroid galling could be observed in all genotypes investigated, but occurred regularly only in a few particular host/P. brassicae combinations. While spindle gall formation was coincident with the expansion of the stele and infection of secondary tissues by P. brassicae, spheroid galls typically had a region of proliferating tissue that corresponded to the secondary cortex and periderm of the healthy plants, with the outer proliferating tissue less infected than the inner portions. The underlying host tissue showed limited secondary tissue development, was largely uninfected, and, where infection occurred, a continuous stele was maintained. An active host defensive reaction, in the form of cell lignification or the hypersensitive response, was not observed, while pathogen resting spores were visible in one longitudinal section of a spheroid gall. These findings suggest that while the proliferation of P. brassicae is restricted in spheroid galls, these structures are not indicative of complete resistance to clubroot.     

Identification and characterization of Agrobacterium spp. isolated from apricot in Serbia

Crown gall, caused by tumorigenic strains of Agrobacterium spp., is considered one of the most important diseases in stone fruit nurseries throughout the world. Since the crown gall disease has not been studied extensively in Serbia for more than 30 years, the objective of this study was to isolate, identify and characterize the bacterium associated with crown gall symptoms on one-year-old apricot trees. Samples were collected from a nursery in central Serbia and subjected to laboratory analysis. Bacteria were isolated from tumour tissue on yeast mannitol agar (YMA) and six gram-negative isolates were selected for further study. PCR assay using primers specific for virD2, virC, ipt and tms2 pathogenicity-associated plasmid genes revealed that isolates harbour Ti plasmids. All studied strains carrying Ti plasmids were classified as nopaline-type based on further molecular analysis. Using a multiplex PCR assay, targeting 23S rRNA gene sequences, and physiological and biochemical tests, five strains were assigned as A. rhizogenes/biovar 2 and the remaining one as A. tumefaciens/biovar 1. Identity of the strains was confirmed by sequence analysis of the 16S rRNA gene. In pathogenicity assay, all six strains caused tumour formation on inoculated carrot root discs, young tomato and sunflower plants.     

Pectobacterium spp. associated with bacterial stem rot syndrome of potato in Canada

Pectobacterium atrosepticum, P. carotovorum subsp. brasiliensis, P. carotovorum subsp. carotovorum, and P. wasabiae were detected in potato stems with blackleg symptoms using species- and subspecies-specific polymerase chain reaction (PCR). The tests included a new assay for P. wasabiae based on the phytase gene sequence. Identification of isolates from diseased stems by biochemical or physiological characterization, PCR, and multi-locus sequence typing (MLST) largely confirmed the PCR detection of Pectobacterium spp. in stem samples. P. atrosepticum was most commonly present but was the sole Pectobacterium sp. detected in only 52% of the diseased stems. P. wasabiae was most frequently present in combination with P. atrosepticum and was the sole Pectobacterium sp. detected in 13% of diseased stems. Pathogenicity of P. wasabiae on potato and its capacity to cause blackleg disease were demonstrated by stem inoculation and its isolation as the sole Pectobacterium sp. from field-grown diseased plants produced from inoculated seed tubers. Incidence of P. carotovorum subsp. brasiliensis was low in diseased stems, and the ability of Canadian strains to cause blackleg in plants grown from inoculated tubers was not confirmed. Canadian isolates of P. carotovorum subsp. brasiliensis differed from Brazilian isolates in diagnostic biochemical tests but conformed to the subspecies in PCR specificity and typing by MLST.     

Pathogenicity of plant and soil isolates of <Emphasis Type="Italic">Phytophthora parasitica</Emphasis> on tomato and pepper

Crown and root rot of tomato and sweet pepper can be caused by Phytophthora parasitica. In this work, 23 P. parasitica isolates from diseased pepper or tomato plants as well as 54 isolates from 23 monocrop tomato soils (from Spain and Chile) and one from a pepper soil were studied for their host–pathogen response. Results show significant host specificity for the isolates from tomato plants and tomato soils (63 of 64 isolates were unable to cause disease in pepper). None of the pepper plant/soil isolates showed pathogenicity on tomato, and only four of 14 reproduced their pathogenicity on pepper. Only one tomato isolate was pathogenic to both Solanaceae species. Two different inoculation protocols were evaluated (substrate irrigation and stem cutting). All isolates which expressed pathogenicity when stem inoculated also did it when root inoculated, but not vice-versa. Therefore, the recommended test protocol for tomato and pepper breeding programmes is that based on root inoculation by irrigation.     

Inhibitory effect of ACC deaminase‐producing bacteria on crown gall formation in tomato plants infected by Agrobacterium tumefaciens or A. vitis

This study showed that various rhizosphere bacteria producing the enzyme 1‐aminocyclopropane‐1‐carboxylate (ACC) deaminase (ACCD), which can degrade ACC, the immediate precursor of ethylene in plants, and thereby lower plant ethylene levels, can act as promising biocontrol agents of pathogenic strains of Agrobacterium tumefaciens and A. vitis. Soaking the roots of tomato (Solanum lycopersicum) seedlings in a suspension of the ACCD‐producing Pseudomonas putida UW4, Burkholderia phytofirmans PsJN or Azospirillum brasilense Cd1843 transformed by plasmid pRKTACC carrying the ACCD‐encoding gene acdS from UW4, significantly reduced the development of tumours on tomato plants injected 4–5 days later with pathogenic Agrobacterium strains via wounds on the plant stem. The fresh mass of tumours formed by plants pretreated with ACCD‐producing strains was typically four‐ to fivefold less than that of tumours formed on control plants inoculated only with a pathogenic Agrobacterium strain. Simultaneously, the level of ethylene evolution per amount of tumour mass on plants pretreated with ACCD‐producing bacteria decreased four to eight times compared with that from tumours formed on control plants or plants pretreated with bacteria deficient in ACCD production. Moreover, transgenic tomato plants expressing a bacterial ACCD were found to be highly resistant to crown gall formation relative to the parental, non‐transformed tomato plants. The results support the hypothesis that ethylene is a crucial factor in Agrobacterium tumour formation, and that ACCD‐produced rhizosphere bacteria may protect plants infected by pathogenic Agrobacteria from crown gall disease.