Diseased Tobacco Plants Research Articles

Analysis of the composition and function of rhizosphere microbial communities in plants with tobacco bacterial wilt disease and healthy plants

ABSTRACT To explore the factors influencing the occurrence of bacterial wilt, the differences in the physicochemical properties, microbial community composition and function between rhizosphere soil of tobacco plants with bacterial wilt and healthy plants in the tobacco planting area of Fuzhou City, Jiangxi Province were analyzed and compared. The results showed that the rhizosphere soil of diseased tobacco exhibited significantly reduced levels of exchangeable potassium, water-soluble potassium, nitrate nitrogen, total nitrogen and pH, in comparison to the rhizosphere soil of healthy plants. Conversely, the available phosphorus content of the rhizosphere soil of diseased tobacco was significantly increased. The amount of Ralstonia solanacearum in soil was negatively correlated with pH, nitrate nitrogen and total nitrogen, and positively correlated with exchangeable potassium and water-soluble potassium. A total of 43 genera were significantly different between the two groups of rhizosphere soil, of which 24 genera were enriched in the rhizosphere of healthy plants, including Ideonella , Rhizophagus , Rhizobacter , Altererythrobacter and Ignavibacterium associated with plant disease resistance, Thermodesulfovibrio , Syntrophorhabdus , Syntrophus , Chlorobium , Hydrogenophaga and Limnohabitans associated with soil sulfur metabolism, as well as Ignavibacterium , Ideonella , Derxia and Azohydromonas associated with soil nitrogen cycling. Kyoto Encyclopedia of Genes and Genomes functional analysis of the unigenes obtained by metagenomic sequencing also showed that the differential unigenes were significantly enriched in the sulfur metabolism pathway. In addition, the rhizosphere soil of diseased tobacco plants exhibited a higher abundance of antibiotic-producing actinomycetes and an increased load of antibiotic resistance genes compared to that of healthy plants. In general, lower pH value, less content of nitrate nitrogen and total nitrogen, and more content of exchangeable potassium and water-soluble potassium could contribute to onset of bacterial wilt. Twenty-four genera, including Ideonella and Rhizophagus , may construct a healthy microecological network in the rhizosphere of tobacco plants. All these factors may interact with each other to control the development of bacterial wilt. This complicated interaction network needs to be explored further. IMPORTANCE Previous studies have mainly focused on the differences in microbial species composition between healthy and diseased soils, but the differences in microbial community functions between two types of soil have not been well characterized. In this study, soil samples in diseased and healthy plant rhizospheres were collected for physicochemical property testing and metagenomic sequencing. We focused on analyzing the differences in physicochemical properties and microbial community functions between these soils, as well as the correlation between these factors and pathogen content. The results of this study provide a theoretical basis for further understanding the occurrence of tobacco bacterial wilt in the field.

Bacterial wilt affects the structure and assembly of microbial communities along the soil-root continuum

BackgroundBeneficial root-associated microbiomes play crucial roles in enhancing plant growth and suppressing pathogenic threats, and their application for defending against pathogens has garnered increasing attention. Nonetheless, the dynamics of microbiome assembly and defense mechanisms during pathogen invasion remain largely unknown. In this study, we aimed to investigate the diversity and assembly of microbial communities within four niches (bulk soils, rhizosphere, rhizoplane, and endosphere) under the influence of the bacterial plant pathogen Ralstonia solanacearum.ResultsOur results revealed that healthy tobacco plants exhibited more diverse community compositions and more robust co-occurrence networks in root-associated niches compared to diseased tobacco plants. Stochastic processes (dispersal limitation and drift), rather than determinism, dominated the assembly processes, with a higher impact of drift observed in diseased plants than in healthy ones. Furthermore, during the invasion of R. solanacearum, the abundance of Fusarium genera, a known potential pathogen of Fusarium wilt, significantly increased in diseased plants. Moreover, the response strategies of the microbiomes to pathogens in diseased and healthy plants diverged. Diseased microbiomes recruited beneficial microbial taxa, such as Streptomyces and Bacilli, to mount defenses against pathogens, with an increased presence of microbial taxa negatively correlated with the pathogen. Conversely, the potential defense strategies varied across niches in healthy plants, with significant enrichments of functional genes related to biofilm formation in the rhizoplane and antibiotic biosynthesis in the endosphere.ConclusionOur study revealed the varied community composition and assembly mechanism of microbial communities between healthy and diseased tobacco plants along the soil-root continuum, providing new insights into niche-specific defense mechanisms against pathogen invasions. These findings may underscore the potential utilization of different functional prebiotics to enhance plants’ ability to fend off pathogens.

First report of Fusarium tricinctum Species complex causing root rot of tobacco in China.

Tobacco (Nicotiana tabacum L.) is an economically important crop in China. In June 2021, a root rot disease was observed on tobacco (cv. Yunyan99) in Lushi, Mianchi, and Luoning counties of western Henan, China. Diseased tobacco plants exhibited wilting with leaf chlorosis and root rot accompanied by purplish to brown vascular discoloration. The symptoms were observed in four surveyed fields, 57 ha in total, and disease incidence ranged from 21 to 56%. Five symptomatic plants with leaf chlorosis and root rot were randomly collected from each field for pathogen isolation. Tissue pieces from diseased roots were surface sterilized in 75% ethanol for 30 s then rinsed with sterile distilled water three times, air dried, and placed onto potato dextrose agar (PDA) medium. Five isolates, SL1, SL2, SL3, LN and KC, were purified by single-spore culturing. On PDA, colonies grew at a rate of 2-5 mm/day and produced abundant cottony, white to pink aerial mycelia and rose pigment on the reverse side of the culture plate. From 7-day-old cultures grown on carnation leaf agar (CLA), macroconidia were straight to subarcuate, with blunt and slightly hooked apical and basal cells, had three to four septa, measured 23.4 to 44.6×3.5 to 4.2 μm (n=30). Cylindrical, napiform or oval microconidia were one to two-celled, measuring 6.3 to 22.9×2.2 to 4.9 μm (n=30). Spherical chlamydospores were intercalary or terminal, in chains. Such characteristics resembled those of the Fusarium tricinctum species comples (FTSC; Batra and Lichtwardt 1962; Leslie and Summerell 2006). To confirm the morphological diagnosis, the genomic DNA of the isolates were extracted, the translation elongation factor 1-alpha (EF-1α), RNA polymerase I largest subunit (RPB1) and second largest subunit (RPB2) genes were amplified with primers EF1/EF2, F5/G2R and 5f2/7cr respectively (O'Donnell et al. 2010), and sequenced. Maximum likelihood analysis was carried out using MEGA 7. Sequences were 97.55% to 100% identical to corresponding DNA sequences of FTSC based on GenBank and Fusarium MLST BLASTn analysis, and deposited in GenBank (ON637268.1-ON637272.1, ON637275.1-ON637279.1, ON637282.1-ON637286.1). Based on the morphological characteristics and phylogenetic analysis, the isolates were identified as F. acuminatum (SL1, SL2, SL3 and LN; Senatore et al. 2021) and F. reticulatum (KC; Moreira et al. 2019). Koch's postulates were conducted to verify the pathogenicity of individual isolates. The four-leaf stage healthy tobacco seedlings (Yunyan99, n=30) were inoculated by pouring 20 mL conidial suspension (1×107 conidia/mL) around the rhizosphere. Control seedlings were inoculated with sterilized water (n=30). All the treatments were maintained under greenhouse conditions with a 12-h light/dark photoperiod at 25±0.5℃ and 70% relative humidity for 30 days. The assay was conducted three times. Foliage chlorosis and root rot were observed on the inoculated tobacco seedlings, whereas the control seedlings remained asymptomatic after 30 days. The pathogens were reisolated from the necrotic tissue from all inoculated seedlings and were identified by sequencing partial EF-1α and RPB2 genes. Fusarium tricinctum species complex are known as an important causal of cereals Fusarium Head Blight (FHB; Laraba, et al. 2022). In China, F. acuminatum can also infect herb plants and fruits, such as Angelica sinensis, Schisandra chinensis (Ma et al. 2022; Shen et al. 2021). To our knowledge, this is the first report of root rot on tobacco caused by FTSC members in China as well as the world. This finding expands the host range known for FTSC and will be helpful for developing effective control strategies of tobacco root rot.

First report of Ludwigia yellow vein Vietnam virus causing leaf curling on tobacco plants in Hainan province, China.

Cigar tobacco (Nicotiana tabacum L.) has been recently introduced into China for various industrial applications. From March 2022, certain symptoms of begomovirus infection, including leaf curling and thickening of veins, were sporadically (disease incidence was approximately 0.2%) observed in several cigar tobacco plantations in numerous counties in Hainan Province, China (Figure 1A). These typical symptoms of begomovirus infection were similar to those caused by the sida leaf curl virus-Hainan (SiLCV-HN) begomovirus and its associated betasatellite, as reported in our previous study on cigar tobacco plants in the same region (Wang et al. 2022). In order to determine whether these symptoms were caused by SiLCV-HN or other begomoviruses, samples of leaves were collected from the diseased tobacco plants for DNA extraction, and the total DNA was extracted for viral metagenomics using an Illumina Sequencing platform at Tiangen Biotech, Beijing. A total of 65711396 filtered reads were obtained, of which 65362322 (99.47%) reads matched to the genome of tobacco. The remaining unmapped 349074 (0.53%) reads were analyzed by BLASTn against the virus Refseq Database of GenBank and subsequently assembled. A total of 8 (5+2+1) enriched contigs of the complete sequence of ludwigia yellow vein Vietnam virus (LuYVVNV) and 9 (8+1) contigs of ludwigia yellow vein virus-associated DNA beta (LuYVB) were finally obtained (Table 1). LuYVVNV belongs to the Begomovirus genus that infects various weeds, including Ludwigia octovalvis and Impatiens balsamina. As far as we know, it was reported earliest on weed in Vietnam (Ha et al. 2008). GenBank contains data pertaining to previously identified isolates of LuYVVNV, and the data revealed that the virus was discovered in Vietnam and the Yunnan province of China currently. However, there are no reports on the infection of crops by LuYVVNV to date. The findings of the present study indicated that LuYVVNV and LuYVB could be responsible for the aforementioned symptoms observed on cigar tobacco. The complete genomes of LuYVVNV and LuYVB were amplified using primer pairs designed based on sequence assembly for viral metagenomics (Table 2). Indeed, two DNA bands with length 2763 bp of LuYVVNV genome and 1348 bp of LuYVB were amplified from leaf samples of diseased tobacco (Figure 1B). The products of polymerase chain reaction (PCR) amplification were analyzed by Sanger sequencing, and the complete nucleotide sequences of LuYVVNV and its associated betasatellite were obtained. Analysis with the BLASTn tool of NCBI revealed that the genome sequence of LuYVVNV isolated from the Hainan province of China had the highest identity of 96.9% to a different isolate of LuYVVNV (GenBank accession number: MN210347.1). These two isolates belong to the same strain, according to the latest revision of Begomovirus taxonomy (Brown et al. 2015). The isolate of LuYVVNV identified in this study was designated as LuYVVNV, Hainan isolate (LuYVVNV-HN, GenBank accession number: OP948731). BLASTn analysis further revealed that the associated betasatellite had the highest sequence identity of 96.9% with an LuYVB (GenBank accession number: AJ965541.1) of a different viral isolate, according to the classification and nomenclature of DNA betasatellites of begomoviruses (Briddon et al. 2008). The sequence of LuYVB obtained herein was therefore designated as LuYVB, Hainan isolate (LuYVB-HN, GenBank accession number: OP948732). The pathogenicity of LuYVVNV-HN and LuYVB-HN was determined using infectious clones that were constructed by ligating two fragments of LuYVVNV-HN or LuYVB-HN to a binary pCAMBIA1300 expression vector, as previously described (Wang et al. 2019). Infectious clones of LuYVVNV-HN, LuYVB-HN, and LuYVVNV-HN plus LuYVB-HN were separately agroinfiltrated into N. benthamiana for determining viral pathogenicity. The typical symptoms of begomovirus infection were observed in N. benthamiana plants inoculated with LuYVVNV-HN alone or LuYVVNV-HN plus LuYVB-HN, and the emerging leaves were mildly or severely down-curled, respectively, at 7 days post inoculation (dpi), with 100% disease incidence (6/6) (Figure 1C). Positive PCR products of the AV1 gene of LuYVVNV-HN were obtained from N. benthamiana plants inoculated with LuYVVNV-HN alone or LuYVVNV-HN plus LuYVB-HN. The βC1 gene of LuYVB-HN was only obtained from N. benthamiana plants co-infected with LuYVVNV-HN and LuYVB-HN (Figure 1D). No symptoms of viral infection were observed in plants individually inoculated with LuYVB-HN, and the results of PCR were negative (Figure 1C and 1D). These findings indicated that the N. benthamiana plants had been successfully inoculated with LuYVVNV-HN, and that LuYVB-HN was incapable of causing infections on its own, but functioned as a helper and enhanced viral pathogenicity. This report is the first to identify isolates of LuYVVNV and LuYVB from cigar tobacco, which is an economically important crop plant. The findings provide insights into the epidemic threat of begomovirus reservoirs in weeds to crop plants, and emphasize the need for monitoring and controlling whitefly-transmitted viral diseases in tobacco plantations worldwide (Ye et al. 2021).

Physiochemical Properties and Microflora of the Rhizosphere Soil of Tobacco Plants with and without Bacterial Wilt

Bacterial wilt is a destructive soilborne disease caused by Ralstonia solanacearum, posing a severe threat to plants in the Solanaceae family. It impacts on tobacco productivity worldwide. This study was conducted to analyze the changes in the soil’s physical and chemical properties, the number of microbes, and the bacterial diversity of the rhizosphere soil before and after the wilt disease. The rhizosphere soil of healthy and diseased tobacco plants was collected from Pucheng, Nanping, Fujian Province, Southern China. The results revealed significant differences in the trends of physical and chemical properties of the soil of healthy and diseased plants. The soil pH, available potassium (K), available phosphorous (P), and organic matter contents (SOM) were lower in the rhizosphere soil for healthy plants than for pre-diseased plants (HW). Only the available P, among all physical and chemical properties in the rhizosphere of diseased plants (HS), was significantly lower than those for pre-diseased plants (HW), changing from 149.59 mg/kg to 59.19 mg/kg. The order of numbers of the three main microbes in the rhizosphere soil for healthy plants (HC) and pre-diseased plants was the following: bacteria > actinomycetes > fungi. The number of actinomycetes in the soil of the diseased tobacco plants increased significantly. A comparison of the rhizosphere soil of diseased and healthy tobacco plants showed that the relative abundance of the bacterial community in the rhizosphere soil of the pathogenic tobacco plants changed significantly. The community diversity was increased, and the Pseudomonadaceae, to which the bacterial pathogen of bacterial wilt belonged, rose to a certain extent. Both pre-diseased and healthy plants showed changes in the physical and chemical properties, microbial quantity, and microbial diversity, thus proving that tobacco disease was closely related to the soil’s ecological environment.

Improved YOLOX-Tiny network for detection of tobacco brown spot disease.

Tobacco brown spot disease caused by Alternaria fungal species is a major threat to tobacco growth and yield. Thus, accurate and rapid detection of tobacco brown spot disease is vital for disease prevention and chemical pesticide inputs. Here, we propose an improved YOLOX-Tiny network, named YOLO-Tobacco, for the detection of tobacco brown spot disease under open-field scenarios. Aiming to excavate valuable disease features and enhance the integration of different levels of features, thereby improving the ability to detect dense disease spots at different scales, we introduced hierarchical mixed-scale units (HMUs) in the neck network for information interaction and feature refinement between channels. Furthermore, in order to enhance the detection of small disease spots and the robustness of the network, we also introduced convolutional block attention modules (CBAMs) into the neck network. As a result, the YOLO-Tobacco network achieved an average precision (AP) of 80.56% on the test set. The AP was 3.22%, 8.99%, and 12.03% higher than that obtained by the classic lightweight detection networks YOLOX-Tiny network, YOLOv5-S network, and YOLOv4-Tiny network, respectively. In addition, the YOLO-Tobacco network also had a fast detection speed of 69 frames per second (FPS). Therefore, the YOLO-Tobacco network satisfies both the advantages of high detection accuracy and fast detection speed. It will likely have a positive impact on early monitoring, disease control, and quality assessment in diseased tobacco plants.

A Duplex PCR Assay for Rapid Detection of Phytophthora nicotianae and Thielaviopsis basicola.

A duplex PCR method was developed for simultaneous detection and identification of tobacco root rot pathogens Phytophthora nicotianae and Thielaviopsis basicola. The specific primers for P. nicotianae were developed based on its internal transcribed spacer (ITS) regions of ribosomal gene, ras gene and hgd gene, while the specific primers for T. basicola were designed based on its ITS regions and β-tubulin gene. The specificity of the primers was determined using isolates of P. nicotianae, T. basicola and control samples. The results showed that the target pathogens could be detected from diseased tobacco plants by a combination of the specific primers. The sensitivity limitation was 100 fg/μl of pure genomic DNA of the pathogens. This new assay can be applied to screen out target pathogens rapidly and reliably in one PCR and will be an important tool for the identification and precise early prediction of these two destructive diseases of tobacco.

Viral Carcinogenesis.

Cancer has been recognized for thousands of years. Egyptians believed that cancer occurred at the will of the gods. Hippocrates believed human disease resulted from an imbalance of the four humors: blood, phlegm, yellow bile, and black bile with cancer being caused by excess black bile. The lymph theory of cancer replaced the humoral theory and the blastema theory replaced the lymph theory. Rudolph Virchow was the first to recognize that cancer cells like all cells came from other cells and believed chronic irritation caused cancer. At the same time there was a belief that trauma caused cancer, though it never evolved after many experiments inducing trauma. The birth of virology occurred in 1892 when Dimitri Ivanofsky demonstrated that diseased tobacco plants remained infective after filtering their sap through a filter that trapped bacteria. Martinus Beijerinck would call the tiny infective agent a virus and both Dimitri Ivanofsky and Marinus Beijerinck would become the fathers of virology. Not to long thereafter, Payton Rous founded the field of tumor virology in 1911 with his discovery of a transmittable sarcoma of chickens by what would come to be called Rous sarcoma virus or RSV for short. The first identified human tumor virus was the Epstein-Barr virus (EBV), named after Tony Epstein and Yvonne Barr who visualized the virus particles in Burkitt's lymphoma cells by electron microscopy in 1965. Since that time, many viruses have been associated with carcinogenesis including the most studied, human papilloma virus associated with cervical carcinoma, many other anogenital carcinomas, and oropharyngeal carcinoma. The World Health Organization currently estimates that approximately 22% of worldwide cancers are attributable to infectious etiologies, of which viral etiologies is estimated at 15-20%. The field of tumor virology/viral carcinogenesis has not only identified viruses as etiologic agents of human cancers, but has also given molecular insights to all human cancers including the oncogene activation and tumor suppressor gene inactivation.

Molecular Identification and Characterization of Tomato zonate spot virus in Tobacco in Guangxi, China.

In Guangxi Province of southwest China, diseases caused by Tospoviruses (family Bunyaviridae) pose a serious threat to tobacco (Nicotiana tobacum) production. During surveys conducted annually at Xinrong Village in Jingxi County from 2008 to 2010, more than 130 ha of fields were found to have 10 to 50% of plants exhibiting symptoms similar to spotted wilt caused by Tomato spotted wilt virus (TSWV). During this period, disease symptoms at similar prevalence and incidence were also found at Fushan, Debao County in most of the cultivars produced in these areas, including Yunyan 85, 87, 92, 97, and K326. Symptoms on tobacco varied but commonly included dwarfing, midrib browning, distorted apical buds, and concentric ringspots that coalesced to form large areas of dead leaf tissue. Mechanical inoculation from diseased tobacco leaves with concentric ringspots back to tobacco cv. Yunyan 85 or 87, resulted in 12 of 16 plants with symptoms similar to those observed in the field. No symptoms on plants developed following inoculation with buffer only. Symptoms found in the field resembled those caused by TSWV. However, testing using TSWV-specific antiserum was shown to be negative by double-antibody sandwich-ELISA (Agdia, Elkhart, IN). Total RNA was extracted from 27 diseased tobacco plants collected from different regions in Guangxi using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. RNA extracts were amplified by reverse transcription (RT)-PCR using the degenerate primers T2740 (ATGGGDATNTTTGATTTCATG) and T3920c (TCATGCTCATSAGRTAAATYTCTCT) designed to target the partial RNA-dependent RNA polymerase (RdRp) sequence of members in the genus Tospovirus (3). Amplification was performed at 42°C for 60 min, followed by 35 cycles of PCR (30 s denaturation at 94°C, 45 s annealing at 55°C, and 30 s extension at 72°C) and a 7-min final extension at 72°C. A PCR product of approximately 1.2 kb was amplified from 21 diseased plants. RT-PCR amplicons were cloned into the pUC19-T Simple Vector (TaKaRa, Dalian, China) and sequenced in both directions. Sequences were assembled and analyzed by DNAStar 5.01 (DNASTAR, Madison, WI). Sequences of representative isolates were deposited in GenBank (Accession Nos. JN020022 to JN020027). The 1.2-kb partial RdRp sequences of these isolates were shown to have 94.4 to 95.3% nucleotide identity and 96.5 to 97.5% amino acids identity to Tomato zonate spot virus (TZSV) (GenBank Accession No. NC_010491) (1). Among these TZSV isolates from Guangxi, the partial RdRp sequences have 98.0 to 99.4% nucleotide identity and 98.8 to 100% amino acids identity with each other. The presence of TZSV was further confirmed in diseased tobacco plants by indirect ELISA using antiserum of TZSV (provided by Prof. Zhongkai Zhang, Agricultural Academy of Yunnan, China). TZSV has been characterized as a novel tospovirus on various hosts including tobacco in Yunnan province (1,2). To our knowledge, this is the first report of TZSV-associated disease on tobacco in Guangxi Province, southwest China. Further work is necessary to study the epidemiology and management of the disease.

Morphological and molecular characterization of Phytophthora glovera sp. nov. from tobacco in Brazil

A root rot disease of cultivated tobacco called yellow stunt has been observed in the burley tobacco production areas of Brazil since the early 1990s. Root infecting fungi and straminipiles were isolated from the roots of diseased tobacco plants, including a semi-papillate, homothallic, slow growing Phytophthora species. Pathogenicity trials confirmed that Phytophthora sp. caused root rot and stunting of burley and flue-cured tobaccos. Morphological characteristics of the asexual and sexual stages of this organism did not match any reported Phytophthora species and were very different from the widely known tobacco black shank pathogen P. nicotianae. Phylogenetic analysis based on sequences of the internal transcribed spacer rDNA, β-tubulin and translation elongation factor 1-α regions indicated that this organism represents a previously unreported Phytophthora species that is significantly supported in clade 2 and most closely related to P. capsici. However P. glovera differs from P. capsici in a number of morphological characters, most significantly P. glovera is homothallic and produces both paragynous and amphigynous antheridia while P. capsici is heterothallic and produces only amphigynous antheridia. In this paper we confirmed pathogenicity of this species on tobacco and describe the morphological and molecular characteristics of Phytophthora glovera sp. nov.

Complete nucleotide sequence and genome organization of a Chinese isolate of Tobacco vein distorting virus

Tobacco bushy top disease is caused by tobacco bushy top virus (TBTV, a member of the genus Umbravirus) which is dependent on tobacco vein-distorting virus (TVDV) to act as a helper virus encapsidating TBTV and enabling its transmission by aphids. Isometric virions from diseased tobacco plants were purified and disease symptoms were reproduced after experimental aphid transmission. The complete genome of TVDV was determined from cloned RT-PCR products derived from viral RNA. It was 5,920 nucleotides (nts) long and had the six major open reading frames (ORFs) typical of a member of the genus Polerovirus. Sequence comparisons showed that it differed significantly from any of the other species in the genus and this was confirmed by phylogenetic analyses of the RdRp and coat protein. SDS-PAGE analysis of purified virions gave two protein bands of about 26 and 59kDa both of which reacted strongly in Western blots with antiserum produced to prokaryotically expressed TVDV CP showing that the two forms of the TVDV CP were the only protein components of the capsid.

Paecilomyces farinosus destroys powdery mildew colonies in detached leaf cultures but not on whole plants

Since 2001, several isolates of Blumeria graminis, the causal agent of cereal powdery mildew, maintained on detached leaves at the John Innes Centre, Norwich, UK, have spontaneously become infected with an unknown filamentous fungus whose mycelia have quickly overgrown the powdery mildew colonies and destroyed them completely. A total of five isolates of the contaminant were obtained and identified as Paecilomyces farinosus based on morphological characteristics and rDNA ITS sequence data. To determine whether these P. farinosus isolates can be considered as biocontrol agents (BCAs) of powdery mildews, we studied the interactions between P. farinosus and the following four powdery mildew species: B. graminis f.sp. hordei infecting barley, Oidium neolycopersici infecting tomato, Golovinomyces orontii infecting tobacco and Podosphaera fusca infecting cucumber. The powdery mildew colonies of all these four powdery mildew species were quickly destroyed by P. farinosus in leaf cultures but neither conidial suspensions nor cell-free culture filtrates of P. farinosus isolates could suppress the spread of powdery mildew infections on diseased barley, tomato, tobacco or cucumber plants in the greenhouse. It is concluded that P. farinosus cannot be considered as a promising BCA of powdery mildew infections although it can destroy powdery mildew colonies in detached leaf cultures and can be a menace during the maintenance of such cultures of cereal, apple, cucurbit and tomato powdery mildew isolates.

A chromosome map of the European stone fruit yellows phytoplasma.

A physical map of the European stone fruit yellows phytoplasma strain GSFY1 chromosome was constructed using PFGE-purified genomic DNA from diseased tobacco and tomato plants. The map was generated with single and double digestions of the chromosome with SmaI, BssHII, ApaI, BamHI and XhoI restriction endonucleases and the fragments were resolved by PFGE. Reciprocal double digestions were used to locate 26 restriction sites on the chromosome. Southern blot analysis was also used to assist in the arrangement of the contiguous restriction fragments obtained. From the restriction fragments generated by double digestion, the circular chromosome was calculated to be approximately 635 kb. Loci of two rRNA operons, the operon containing the tuf gene, genes encoding an immunodominant membrane protein and a putative nitroreductase, and randomly cloned DNA fragments IH184 and AT67 were placed on the map. Digestion of chromosomal DNA of strain GSFY1 with MluI gave a complex restriction pattern, suggesting that this isolate consists of a population with heterogeneity with respect to MluI restriction sites. The GSFY1 physical map was different from that of the closely related apple proliferation phytoplasma but the genetic arrangement was similar.

Physical map of the chromosome of the apple proliferation phytoplasma.

A physical map of the apple proliferation phytoplasma strain AT chromosome was constructed from genomic DNA extracted from diseased tobacco plants. The map was generated with single and double digestions of the chromosome with BssHII, SmaI, MluI, and ApaI restriction endonucleases and resolving the fragments by pulsed-field gel electrophoresis. Partial digestion and Southern blot analysis were used to assist in the arrangement of the 14 contiguous restriction fragments obtained. From the restriction fragments generated by double digestions, the size of the circular chromosome was calculated to be approximately 645 kb. Locations of the two rRNA operons, the operon including the fus and tuf genes, and three other genes were placed on the map. Genome sizes and BssHII restriction profiles of apple proliferation strain AP15 and the pear decline and European stone fruit yellows phytoplasmas were different from that of strain AT.

A reappraisal of the contribution of Friedrich Loeffler to the development of the modern concept of virus.

Most virology textbooks, with the exception of the Afterword of A. J. Levine’s chapter in the third edition of Field’s Virology (1996), emphasize the contribution of Martinus Beijerinck (1898) to the birth of the modern concept of virus and they refer to Friedrich Loeffler (1898) only because he was the first to have shown the filterability of the animal virus Foot and mouth disease virus. Following Adolf Mayer [9] who first described the mosaic disease of tobacco and demonstrated that the sap of diseased tobacco plants was infectious, Dimitri Ivanowsky (1896) showed that such saps remained infectious after filtering through Chamberland filters. Beijerinck confirmed Ivanowsky’s observations and further showed that the infectious agent could diffuse through several millimeters of an agar gel, from which he concluded that the agent could not be a bacteria. Beijerinck also showed that the infectivity of sap remained constant during serial infections of plants, providing evidence that the agent could not be a toxin, since it was able to replicate itself in living organisms. The relative contributions of Mayer, Ivanowsky and Beijerinck have been extensively discussed by Bos [2]. Beijerinck was the first to use the term virus for such an infectious agent. He was, however, convinced that the agent was a liquid (or a solute: mentioned p. 6, line 16, in his 1898 article). In the title of his article he therefore named it: contagium vivum fluidum, or contagious living liquid. Beijerinck thought his interpretation was confirmed by the observation that the sap filtered through Chamberland candles was less infectious than unfiltered sap. He explained this reduction as resulting from adsorption of the agent to the surface of the filter, mainly at the beginning of the filtration. In a control experiment he found that the less diffusible (granulase) of two enzymes present in malt extracts was more strongly retained in Chamberland filters than the more diffusible enzyme (maltase), until saturation was reached. In a footnote of his 1898 paper (see Appendix I) he stated that he could not agree with Loeffler’s conclusion that the agent of foot-and-mouth disease was a particle and not a liquid. Friedrich Loeffler and Paul Frosch had been appointed in 1886 by the Government in Berlin to investigate the cause of foot-and-mouth disease and to find a way to protect cattle against the disease. The conclusions of the investigating Committee are described in two articles published a few months before the appearence of Beijerinck’s paper (Reports 1, 2 and 3: Loeffler and Frosch, 1898; Report 4: Loeffler, 1898; this latter article was cited by

Characterization and vector relation of a serologically distinct isolate of tobacco rattle tobravirus (TRV) transmitted byTrichodorus similis in northern Greece

A virus isolated from diseased tobacco plants growing in Macedonia, northern Greece, had host range and physico-chemical properties typical of a tobravirus. Although it was serologically unrelated to any of the ten tobravirus isolates tested, it reacted in spot hybridization tests with a probe derived from RNA-1 of tobacco rattle virus (TRV) strain SYM. Therefore, the isolate probably represents a previously undescribed serotype of TRV. Male, female and juvenileTrichodorus similis nematodes recovered from the rhizosphere of the diseased tobacco plants transmitted TRV in each of three laboratory experiments. In two of these experiments 50% and 54%, respectively, of the nematodes transmitted virus toPetunia hybrida bait plants, whereas only 18% transmitted virus toNicotiana tabacum plants in a third test. Ultrathin sections of the feeding apparatus of individual nematodes, which had transmitted virus, were examined by electron microscopy. Virus particles were observed, retained as a monolayer in the apices of the oesophageal lumen and as a group of particles within a matrix in the open part of the lumen.

5402786 Magneto-acoustic resonance imaging

Cancer has been recognized for thousands of years. Egyptians believed that cancer occurred at the will of the gods. Hippocrates believed human disease resulted from an imbalance of the four humors: blood, phlegm, yellow bile, and black bile with cancer being caused by excess black bile. The lymph theory of cancer replaced the humoral theory and the blastema theory replaced the lymph theory. Rudolph Virchow was the first to recognize that cancer cells like all cells came from other cells and believed chronic irritation caused cancer. At the same time there was a belief that trauma caused cancer, though it never evolved after many experiments inducing trauma. The birth of virology occurred in 1892 when Dimitri Ivanofsky demonstrated that diseased tobacco plants remained infective after filtering their sap through a filter that trapped bacteria. Martinus Beijerinck would call the tiny infective agent a virus and both Dimitri Ivanofsky and Marinus Beijerinck would become the fathers of virology. Not to long thereafter, Payton Rous founded the field of tumor virology in 1911 with his discovery of a transmittable sarcoma of chickens by what would come to be called Rous sarcoma virus or RSV for short. The first identified human tumor virus was the Epstein–Barr virus (EBV), named after Tony Epstein and Yvonne Barr who visualized the virus particles in Burkitt's lymphoma cells by electron microscopy in 1965. Since that time, many viruses have been associated with carcinogenesis including the most studied, human papilloma virus associated with cervical carcinoma, many other anogenital carcinomas, and oropharyngeal carcinoma. The World Health Organization currently estimates that approximately 22% of worldwide cancers are attributable to infectious etiologies, of which viral etiologies is estimated at 15–20%. The field of tumor virology/viral carcinogenesis has not only identified viruses as etiologic agents of human cancers, but has also given molecular insights to all human cancers including the oncogene activation and tumor suppressor gene inactivation.

Strains of Cucumber Mosaic Virus Isolated from Tobacco Plants

A new strain of cucumber mosaic virus (CMV-YM) was isolated from tobacco plant (cv. Bright Yellow) collected in Kagawa Prefecture, Shikoku Island, Japan, in 1960. In 1969 the occurrence of the same strain was also observed in some districts in Okayama Prefecture. The diseased tobacco plants showed only yellow mottle symptoms without malformation and necrotic lesions, and little retard of plant growth.By the sap inoculation, the virus induced brilliant yellow mottle on seedlings of some cultivars of tobacco, Nicotiana glutinosa, N. sylvestris and red pepper (Capsicum annuum). The symptoms on these inoculated host plants are characterized by the lack of malformation, distortion and necrosis. The virus caused mild green mottle in Zinnia elegans, local and systemic necrotic lesions in sesame, mild yellow mottle in cucumber and necrotic local lesions on inoculated leaves of cowpea, Chenopodium amaranticolor and C. quinoa. Physalis floridana and spinach showed green mottle and a little malformation. The strain can be differenciated from other strains of CMV, viz. ordinary (CMV-O), yellow (CMV-Y), mild (CMV-C) and legume (CMV-LE) strains, which were obtained from field tobacco in Japan, by the reactions of some differencial hosts, such as tobacco, N. glutinosa, sesame, cowpea and zinnia.Thermal inactivation point of crude juice extracted from infected tobacco leaves was 55-60°C for 10 minutes. Dilution end point was around 1:10, 000 with phosphate buffer (0.1M, pH 7.0). Longevity in vitro was 2-3 days at 25°C. Phenol extracts from infected tobacco leaves were more infectious than control buffer-homogenate, similarly to those of the other strains. Cross protection against other strains including necrotic strain (CMV-N), in tobacco, cowpea and zinnia was completely positive. CMV-C strain gave incomplete protection against the other strains (Tomaru et al., 1967), but it gave complete protection against the present virus, suggesting that the latter is more closely related to CMV-C. It needs, however, further investigations to determine the relationship between these two strains. Serological positive reactions were observed between the present virus and CMV-Y antiserum, and antiserum of the present virus and other 5 strains in agar-gel diffusion and microdroplet-precipitin tests. Electron microscopy of the purified virus showed spherical or polyhedral particles with a diameter of about 30mμ. The periodic change in the virus concentration in top leaves of infected tobacco plant was almost similar to the case in CMV-O.For the strain described here the authors propose the name yellow mild mottle strain of CMV (CMV-YM).

Virus-induced nucleolar abnormalities in tomato.

IN view of current opinions on the nature of virus particles1, recent work carried out in this department on the effect of the ‘aspermy’ virus upon nuclear, and especially nucleolar, behaviour in plant cells may have a special significance. Caldwell2 reported prophase collapse in diseased pollen- and embryo-sac mother cells in tomato, accompanied by peculiarities of nucleolar behaviour, and Wilkinson3 has observed similar meiotic abnormalities in Nicotiana glutinosa. Recent examination of somatic divisions in diseased tomato and tobacco plants has revealed the occurrence of widespread nucleolar and other abnormalities, some of which recall the aberrations regarded as typical of the cell-divisions found in malignant animal tissue; for example, arrested metaphase, collapse at metaphase, spindle malformation and breakdown, tendency to form giant nuclei, etc. In particular, abnormal persistence of the nucleolar material, and sometimes an apparent multiplication of nucleolar bodies during the mitotic cycle, have been very striking. Thus, in dividing cells of root tips obtained from tomato shoots heavily infected with the ‘aspermy’ virus, the nucleolar material, instead of dispersing during prophase, persists through anaphase in the form of one or more prominent and somewhat elongated vesicles. The accompanying photograph shows a typical dividing nucleus at early anaphase, with the nucleolar vesicle in a central position upon the somewhat distorted spindle; incidentally, in the cell above can be seen an example of metaphase collapse. Similar results have been obtained in tobacco species infected with the same virus.

Respiration of the tissue of mosaic diseased tobacco plants

1. Using the WARBURG'S respiration manometer, the respiration of the tissues of tobacco plants infected by ordinary mosaic virus was compared with that of the healthy plants.2. In the diseased leaf tissues, the amounts of oxygen consumption and carbon dioxide production are greater than the healthy one, but the repiratory quotient shows the similar value of about 0.94. Anaerobic respiration is also stronger in the diseased leaves. The increase of respiration in the diseased leaves is markedly accelerated in older infected leaves.3. In the root tissues, the consumed oxygen is the same degree in the diseased and healthy plants. The carbon dioxide production both in the aerobic and anaerodic conditions is very low in the mosaic plants. It is considored that the low value of respiratory quotient is due to the imcomplete oxidation in the process of the aerobic respiration in diseased plants.4. The expressed juice of the mosaic plants showed no detectable gas exchange.5. The expressed juice of diseased leaves does not affect the respiratory rate pollen of Lilium longiflorum.