Diseased Plant Material Research Articles

Pruning and cypress canker in New Zealand

In New Zealand, the planting of cypresses is hindered by cypress canker disease caused by Seiridium cardinale (W.W. Wagener) B. Sutton & I.A.S. Gibson and S. cupressi (Guba) Boesew.). Pruning is widely practiced in established plantations to improve timber quality, although knowledge of its effect on disease incidence remains imperfect. Stubs of freshly cut branches on 275 trees of Cupressus macrocarpa Gordon & Leyland were inoculated with an isolate of either S. cardinale or S. cupressi to investigate the potential for pruning wounds to act as infection courts. In total, 75% of stubs inoculated at the edge, and 60% of those inoculated at the centre, became infected and produced stem cankers after 1 year, whereas cankers were formed on only 2% of non-inoculated control stubs. Incidence of cankers differed significantly between isolates. A second experiment was conducted at the same site 2 years later when stand infestation had increased, this time without inoculating but instead relying on ambient field inoculum. Stem cankers were produced on 11% of stubs on 162 trees, as a result of natural infection following pruning during periods of light rain in spring and summer. This investigation confirmed the ability of Seiridium species to invade through fresh pruning wounds on C. macrocarpa, and strengthens the recommendation to remove diseased plant material early, to maintain a low level of natural-spore inoculum within stands. It also indicates that pruning during wet weather should be avoided. Cleaning or disinfecting implements among trees may also be appropriate when conducting silvicultural operations in diseased plantations.

Biological control of necrotrophic plant pathogenic fungi: Tomato plants as a model

Biocontrol agent, Bacillus subtilis, was screened in a laboratory experiment against 6 necrotrophic fungi. These fungi were isolated from diseased plant materials. This biocontrol agent exhibited different potentials. It was highly effective against Fusarium oxysporum f. sp. lycopersici which causes tomato wilt and Fusarium oxysporum f. sp. fabae which causes broad bean wilt. It inhibited radial growth (RGI) by more than 75 %. However, it was less effective against Phytophthora infestans (64.7 % RGI), Botrytis cinerea (58.3 % RGI), Sclerotinia sclerotiorum (57.4 % RGI) Rhizoctonia solani (42.7 % RGI). When B. subtilis was applied to Tomato field, it exhibited different efficiency in suppressing Fusarium wilt. Time of implementation was critical for the efficacy of B. subtilis. It strongly suppressed wilt when applied 7 days before transplanting, by showing 72.7, healthy plant percentage (HP), followed by application at cotyledon stage (69.4 % HP). Salicylic acid was comparable to B. subtilis in protecting tomato against vascular wilt (68.3 % HP). Different mechanisms may be involved in the activity of B. subtilis in field. According to the laboratory test, diffusible suppressive substances as well as lytic enzymes may be produced by this biocontrol agent.Egyptian Journal of Biotechnology Vol. 25 (1) 2007: pp. 1-12

Armillaria Species Infesting Vineyards in Northwestern Spain

Twelve vineyards in northwestern Spain were studied to assess the incidence of white root rot during 1995 and 1997. In both years, diseased plant material was collected and the Armillaria species responsible was identified on the basis of compatibility testing. Subsequently, restriction fragment length polymorphism (RFLP) analysis of the intergenic spacer region of the nuclear ribosomal RNA gene cluster was used to identify Armillaria species in another 45 symptomatic samples submitted for diagnosis from this area. During 1995, 9 of the 12 vineyards showed white root rot, although in eight cases less than 2% of plants were affected; in the remaining vineyard 17% of plants were diseased. During 1997, 10 of the 12 vineyards were affected and three showed a marked increase in the number of plants with white root rot (to 43%, in the vineyard in which 17% were affected in 1995). The compatibility method detected Armillaria mellea in samples from 10 of the 12 vineyards, and Armillaria gallica in samples from two vineyards. RFLP–PCR analysis detected four restriction patterns corresponding to A. mellea, A. gallica and Armillaria cepistipes: patterns mel 1 (A. mellea, 71% of samples), mel 2 (A. mellea, 18%), gal 1 (A. gallica, 9%), and cep 1 (A. cepistipes, 2%, i.e. a single sample). This is the first report of A. gallica and A. cepistipes infecting Vitis spp. The presence of these two Armillaria species may be related to the fact that the vineyards from which they were isolated were located on cleared forestry sites.

Molecular Detection of Phytophthora ramorum, the Causal Agent of Sudden Oak Death in California, and Two Additional Species Commonly Recovered from Diseased Plant Material

ABSTRACT Sudden oak death is a disease currently devastating forest ecosystems in several coastal areas of California. The pathogen causing this is Phy-tophthora ramorum, although species such as P. nemorosa and P. pseudo-syringae often are recovered from symptomatic plants as well. A molecular marker system was developed based on mitochondrial sequences of the cox I and II genes for detection of Phytophthora spp. in general, and P. ramorum, P. nemorosa, and P. pseudosyringae in particular. The first-round multiplex amplification contained two primer pairs, one for amplification of plant sequences to serve as an internal control to ensure that extracted DNA was of sufficient quality to allow for polymerase chain reaction (PCR) amplification and the other specific for amplification of sequences from Phytophthora spp. The plant primers amplified the desired amplicon size in the 29 plant species tested and did not interfere with amplification by the Phytophthora genus-specific primer pair. Using DNA from purified cultures, the Phytophthora genus-specific primer pair amplified a fragment diagnostic for the genus from all 45 Phytophthora spp. evaluated, although the efficiency of amplification was lower for P. lateralis and P. sojae than for the other species. The genus-specific primer pair did not amplify sequences from the 30 Pythium spp. tested or from 29 plant species, although occasional faint bands were observed for several additional plant species. With the exception of one plant species, the resulting amplicons were smaller than the Phytophthora genus-specific amplicon. The products of the first-round amplification were diluted and amplified with primer pairs nested within the genus-specific amplicon that were specific for either P. ramorum, P. nemorosa, or P. pseudo-syringae. These species-specific primers amplified the target sequence from all isolates of the pathogens under evaluation; for P. ramorum, this included 24 isolates from California, Germany, and the Netherlands. Using purified pathogen DNA, the limit of detection for P. ramorum using this marker system was approximately 2.0 fg of total DNA. However, when this DNA was spiked with DNA from healthy plant tissue extracted with a commercial miniprep procedure, the sensitivity of detection was reduced by 100- to 1,000-fold, depending on the plant species. This marker system was validated with DNA extracted from naturally infected plant samples collected from the field by comparing the sequence of the Phytophthora genus-specific amplicon, morphological identification of cultures recovered from the same lesions and, for P. ramorum, amplification with a previously published rDNA internal transcribed spacer species-specific primer pair. Results were compared and validated with three different brands of thermal cyclers in two different laboratories to provide information about how the described PCR assay performs under different laboratory conditions. The specificity of the Phytophthora genus-specific primers suggests that they will have utility for pathogen detection in other Phytophthora pathosystems.

ELECTRON MICROSCOPIC EVIDENCE FOR PURIFICATION OF COCONUT ROOT (WILT) DISEASE PHYTOPLASMA

Root (wilt) disease phytoplasma was purified from diseased coconut tissues using discontinuous Percoll gradient centrifugation method. Crude sap prepared from coconut was layered on a discontinuous Percoll gradient of 15, 30, 50 and 60%(v/v). After centrifugation at 20,000 g for 30 min, the turbid fraction formed on the top of 30% gradient in the diseased plant material was recovered, processed and fixed for electron microscopy. Electron microscopic examination of sections prepared from purified preparation of diseased plant material showed typical cells of root (wilt) phytoplasma with heterogeneous sizes and more or less spherical shape that are similar to those found in sieve elements of diseased tissues and salivary glands of infective (viruliforms) insect vectors. These purified phytoplasma bodies showed trilaminar membrane with internal materials of ribosome granules and DNA fibrils. However, the yield in terms of number of cells was fewer and in addition to intact bodies, free membranes and empty bodies lacking internal contents also were observed.

Obituary

Obituary

First report of Phytophthora nicotianae associated with Eucalyptus die‐back in South Africa

Eucalyptus species, hybrids and clones are planted commercially in South Africa for the production of pulp and paper and various other forest products. However, some species such as E. smithii are susceptible to root and collar rot associated with Phytophthora cinnamomi (Linde et al., 1999). Since 1999, P. nicotianae (synonym, P.n. var. parasitica), not P. cinnamomi, has been recovered from dead and dying Eucalyptus trees such as E. macarthurii and E. smithii. Phytophthora nicotianae is a soilborne pathogen often isolated from forest soils (Shearer et al., 1988). In South Africa it is well known as the causal agent of black butt on Acacia mearnsii (Roux & Wingfield, 1997). In the field, 1-year-old infected trees had typical symptoms of Phytophthora root and collar rot. First disease symptoms included chlorosis of the leaves as well as gum exudation through the cankers on the tree collar. As the disease progressed, the trees usually wilted and died due to girdling. Disease symptoms were reproduced on field-grown trees inoculated with different P. nicotianae isolates. Necrotic lesions with gum exudates were observed above the inoculation point, 30 days after inoculation. Subsequent to the discovery of the root disease caused by P. nicotianae, several recently established E. smithii plantations in the KwaZulu-Natal Province of South Africa were surveyed. P. nicotianae was recovered from 45% of soil samples and 60% of diseased plant material. P. cinnamomi was recovered from only 5% of soil samples and 12% of diseased plant material. Phytophthora isolates from soil and diseased material were identified using morphological features (Stamps et al., 1990). The A1 and A2 mating types were determined by pairing P. nicotianae isolates obtained with known tester strains using the method described by Erwin & Ribeiro (1996). Oospores were produced following pairing with tester isolates and all isolates proved to be of the A2 mating type. Isolates have been deposited into the culture collection of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria. Phytophthora nicotianae, rather than P. cinnamomi, is emerging as a more serious threat to a number of cold-tolerant Eucalyptus spp. in South Africa. Since 1999 P. nicotianae has been recovered from dead and dying Eucalyptus species such as E. fastigata, E. elata, E. macarthurii, E. nites, E. dunii and E. smithii.

A New Disease of Flowering Dogwood Caused by Colletotrichum acutatum.

In 1992 symptoms consisting of twig dieback, canker-like deformities, and often, mortality of all sizes of container-grown flowering dogwood trees (Cornus florida L.) was frequently observed in northern Florida where nursery production of dogwood is concentrated. Twigs, branches, growing points, and portions of main trunks up to 3 cm in diameter were killed. On affected portions, leaves drooped downward but did not wilt severely; they later became silvery, gray-green and eventually died but did not drop. Affected trees produced flower buds, but the flower buds did not open. Many trees died and others were destroyed because of unsightly dead limbs and deformed trunks. The inciting pathogen was not satisfactorily determined. After 1994, disease incidence subsided, but in 1999, it reappeared and caused serious economic damage. In 1999, we followed the disease from propagation through several stages of nursery production, including growth in large containers. A Colletotrichum species was consistently isolated from acervuli produced in abundance near the soil line on diseased, rooted cuttings and small plants. The same fungus was consistently isolated from acervuli produced on diseased leaves and twigs of larger plants and small trees. Conidia of the isolated pathogen were used to inoculate small dogwood trees kept at 100% RH for 2 days then grown in a greenhouse. Within 23 weeks, a slowly developing leaf spot was produced on inoculated plants. After 3 months, numerous acervuli were produced on inoculated leaves and on adjacent small twigs from which the same pathogen was consistently reisolated. The symptoms and pathogen were not consistent with descriptions of anthracnose incited by Discula destructiva. On potato-dextrose agar (PDA), spore masses were bright red-orange on lawn plates inoculated with large numbers of conidia. Conidiomata did not produce setae in culture nor did acervuli on diseased plant material. On PDA, mycelial growth was tufted and pale-gray. The reverse side of colonies was buff to cream-colored or pale-gray to tan but never dark. With age, a pale pink or orange-pink pigment often formed within the agar media (1,3). When produced on PDA, most conidia were elliptical and elongated with abruptly tapering ends. An average of 18% had both ends slightly rounded; none had only one end abruptly tapered or rounded. Conidia measured 15.3 × 4.78 μm; the length/width ratio was 3.20 (1,3). The teleomorph was not found on diseased plant material or in culture. Analysis of DNA by polymerase chain reaction (PCR) using the CaInt2-ITS4 and CgInt-ITS4 primer pairs (1,2) and by comparison of PCR products with those produced by C. acutatum pathotypes isolated from leatherleaf fern, lime, post-bloom fruit drop-affected Navel orange fruit, and strawberry fruit, and with isolates of C. gleosporiodes from citrus, Camellia, Nandina, holly, and strawberry indicated that the fungus was C. acutatum. The amplified PCR product (approximately 490 bp) obtained from all dogwood isolates using primers CaInt2 and ITS4 was consistent with the size of product expected from C. acutatum. No products were produced with the CgInt-ITS4 primer pair. Based on the morphology of conidia, growth in culture and PCR results, the pathogen was identified as C. acutatum and represents the first report of this pathogen on flowering dogwood.

First Report of Leaf and Stem Rust Caused by Puccinia sherardiana on Sphaeralcea grossulariaefolia in North America and S. munroana in Wyoming.

Diseased samples of globe mallow, Sphaeralcea grossulariaefolia and S. munroana, were submitted by an ornamental seed producer in Wyoming to our Extension Plant Pathology Laboratory in July 1997. Dark brown, amphigenous telia surrounded by chlorotic halos were present on both foliage and stems. Mean teliospore dimensions observed were 30.8 × 44.8 μm. The teliospores germinated readily on water agar at 20°C and formed basidiospores within 24 h. Aecia and uredinia were not found. Based on characteristics mentioned above, this fungus was identified as Puccinia sherardiana Körn (1). This microcyclic rust was previously described on 12 other Sphaeralcea spp. plus other plant species in the Malvaceae family (1,2). Stem and foliar symptoms were reproduced in a greenhouse on 8-week-old plants of S. grossulariaefolia and S. munroana. These plants were inoculated with teliospores removed from the original diseased plant material. Immediately after inoculation, plants were misted and placed in plastic bags and incubated for 36 h at 100% relative humidity and 20°C. Plants continued growth with natural lighting and with day and night temperatures of 20 and 15°C, respectively. Symptoms developed within 12 days with initial telia rupturing the host epidermis 13 days after inoculation. Telia were examined microscopically to complete Koch's postulates.

Viruses in Marine Brown Algae

Virus-like particles have been repeatedly reported from multicellular marine red and brown algae, and the release of viruses from infected algal hosts into the surrounding sea water also contributes to the number of virus-like particles in coastal waters. It has become apparent that they are intimately and unseparably connected with the biology of their host organisms. This chapter reviews investigations on this newly discovered group of brown algal viruses. Axenic algal clones with desired growth properties are selected and grown in large quantities in laboratories for anlaysis. In experiments, glass beads are used in a Braun homogenizer to disrupt fresh diseased plant material. After removal of cellular debris by low-speed centrifugation, viral material was precipitated in polyethylene glycol and further purified by equilibrium centrifugation in cesium chloride gradients. The virus particles in these preparations were morphologically less complex than those seen in thin sections through infected cells. Pulsed-field agarose gel electrophoresis (PFGE) is used to determine the size of the viral genome. Pulsed-field gel electrophoresis demonstrated that in addition to EsV, other brown algal viruses also possess large double-stranded DNA as genomes, although their sizes differ significantly among species. On the basis of observations, the interesting possibility remains that brown algal viruses can serve in some cases as carriers for the transmission of foreign genetic information.

Survival of Colletotrichum acutatum in Soil and Plant Debris of Leatherleaf Fern.

Leatherleaf fern anthracnose is incited by a new pathotype of Colletotrichum acutatum. Fern anthracnose appeared in Florida in 1993, has spread rapidly, and produces losses that approach 100% in some ferneries. To help develop recommendations for control and management of fern anthracnose, the survival of the pathogen in soil and diseased plant material and on clothing was investigated in laboratory and field experiments. Survival of conidia, sclerotia, and conidia in infected leaf debris was studied in soil maintained at five soil moisture levels. Survival of conidia and sclerotia declined rapidly under moist conditions (≥12% moisture, vol/wt), but under dry conditions, viable conidia could be detected up to 12 months after incorporation into soil. Similar results were obtained in field experiments in which infected leaf debris was buried in soil. Viable conidia were recovered for up to 3 months in leaf debris stored under laboratory conditions. Conidia applied to denim fabric were recovered up to 5 weeks after application. This indicated that contaminated clothing could transport conidia effectively within and between ferneries. Inoculating ferns with dilutions of conidia demonstrated that at least 100 conidia per ml is required to infect and colonize leatherleaf fern leaves. This threshold provides an estimate of levels of conidia populations likely to result in disease and levels that sanitation or cultural practices must exceed to be effective management methods.

Survival, Germinability and Infectivity of Oospores of Peronospora viciae f.sp. fabae

AbstractA series of experiments was conducted to germinate oospores of Peronospora viciae f.sp. fabae. With rare exceptions, dry‐stored oospores did not germinate in water nor did they infect faba bean seedlings in soil. Long‐term storage, pre‐treatment with KMnO4 or addition of nutrients to the medium did not induce germination. Survival and infectivity of dry‐stored oospores were compared to those of oospores incorporated in a silt loam and a loamy sand soil in the field during 21–22 months. Under dry conditions, the percentage of living oospores did not change as determined by the vital stain tetrazolium bromide. In soil, less than 2% of the oospores had survived after 21 months. Infectivity of oospores was determined by a bioassay 17 and 21 months after oospores had been incorporated in soil. Diseased seedlings were obtained after inoculation of faba bean seeds with oospores extracted from the soil but not with the drystored ones. Soil samples from two field plots naturally infested with oospores 2 and 3 years before the bioassay were infective. Oospores collected with diseased plant material on one of these plots and subsequently stored dry for 3 years were not infective. The results suggested that oospores need a period of natural weathering to become germinable and infective.

INTRA-NURSERY SPREAD OF PLANT PATHOGENS: A CASE FOR EXCLUSION AND SANITATION AS DISEASE CONTROL METHODOLOGIES

Four novel and five commonly occurring diseases of ornamental nursery stock were evaluated for patterns of dissemination and rapidity of movement within a commercial nursery. Newly acquired but infected nursery stock provided a readily available inoculum source. Dissemination, pathogen movement, and disease development were positively correlated to minimal plant proximities, overhead irrigation, and communal root or soil environments. Water containment and recycling systems allowed movement of waterborne pathogens between plants on the same bench, in the same row, or on contiguous sheets of plastic or landscape fabric. Diseased plants located above uninfected stock or upstream or inside overhead irrigation systems provided a source for rapid aerial spread of conidia. Detached diseased plant parts provided rapid physical movement of pathogens and disease developed despite applications of fungicides. Exclusion of diseased plant materials accompanied by rigorous sanitation offer important means of limiting pathogen movement within the nursery.

DNA probes for the detection of plant pathogenic bacteria

During the last few years there has been an increasing interest in exploiting the potential of DNA probes for identification and detection of phytopathogenic bacteria and there are now sufficient reports using representatives of the main relevant genera to show that DNA probes are of unequivocal value in identification. Detection of phytopathogenic bacteria is required under two main sets of circumstances: for diagnosis of diseased plant material and for plant hygiene purposes. The major difference is the demand for sensitivity and thus different DNA probe assays are relevance for the two conditions. Dot-blot assays are simple but not very sensitive. On the other hand, assays based on the polymerase chain reaction can be extremely sensitive. A common problem for many applications appears to be a negative influence on the assays from plant debris and/or background microflora. There are, however, several possibilities to counteract these effects. In this review, we discuss the recent progress made in this field.

Thielaviopsis Basicola, a New Record on Cotton in Australia.

Pale-green, unthrifty cotton plants featuring a cylindrical black layer of diseased tissue in the swollen crown region of the stem, and stunted cotton seedlings with blackened root systems were collected from commercial crops in north-western New South Wales. These symptoms are similar to those ascribed to Thielaviopsis basicola on cotton in the USA. Attempts to isolate the causal agent with standard media failed because of contamination. Fresh carrot segments were successfully used to isolate T. basicola from diseased plant material. This constitutes the first record of T. basicola on cotton in Australia.

THE POSSIBLE APPLICATION OF MONOCLONAL ANTIBODIES IN ELISA FOR DETECTION OF TULIP BREAKING VIRUS

Hybridomas to purified tulip breaking virus (TBV) were produced by H.T. Hsu (American Type Culture Collection). The cell lines were selected in ELISA by a direct coating of the purified virus to the plates. Ascites, containing monoclonal antibodies to TBV, were tested on their activity to other purified viruses of bulbous crops. The results show that ascites react well with purified TBV, and some of them with other, purified, potyviruses as well. Also, the ascites were tested for their use in the detection of TBV in plant material, in comparison to polyclonal antibodies. The antibodies were used for indirect or direct detection, after a direct or indirect coating with mono- or polyclonal antibodies. With purified TBV, practically all monoclonal antibodies reacted better with a direct coating of the virus than with an indirect coating using polyclonal antibodies. A direct coating with diseased plant material did not yield high ELISA values. A coating with monoclonal antibodies derived from one cell line hardly gave any ELISA values, neither for purified virus, nor for diseased material. Therefore, for the detection of TBV in plant material polyclonal antibodies are still preferred to monoclonal antibodies.

Mycoplasma-like Organisms from Plants with 'Yellows' Diseases Lack a Spiroplasma-specific Antigen

An antigen which is specific to Spiroplasma and was unaffected by changes in cell morphology was used to demonstrate the presence of spiroplasmas in diseased plant material. The same antigen could not be detected in plants infected with eight different ‘yellows’ diseases with which non-cultivable mycoplasma-like organisms were associated.

Microscopy and image analysis in the study of plant pathogens and the application of pesticides

AbstractProblems associated with the preparation of diseased plant material for transmission electron microscopy (TEM) and the uses of TEM to meet the specific needs of plant virologists are discussed. Methods of collecting spray droplets for analysis by an image‐analysing computer method are described.

Negatively stained mycoplasmas: Fact or artifact?

Structures generally considered as characteristic mycoplasma-like bodies can be revealed by the electron microscopy of phosphotungstic acid (PTA)-stained extracts prepared from plants infected with the agents of aster yellows and corn stunt disease. However, negative staining of extracts from healthy plants of several species revealed morphologically similar “mycoplasma-like” bodies. That these bodies are preparation and staining artifacts is shown by the observation that no such structures can be found in either the diseased or healthy plant material treated with osmium tetroxide before staining with PTA. Artifacts similar to those seen in the plant material can be produced also by the negative staining of HeLa cell preparations. These findings stress the shortcomings of the negative staining techniques as applied to characterization and identification of mycoplasma microorganisms. Morphological characterization of presumptive plant mycoplasmas based on thin section techniques avoids the pitfalls of negative staining.

Ethyl acetate produced by Ceratocystis paradoxa and C. adiposum and its role in the inhibition of the germination of sugarcane buds

A phytotoxic substance, identified as ethyl acetate by gas chromatography and physical properties, was isolated from the sugarcane tissue infected by Ceratocystis paradoxa and C. adiposum. Ethyl acetate in the diseased tissue could reach 1% by volume and was sufficient to inhibit the germination of sugarcane buds. The saturated vapor of ethyl acetate could kill seed cane buds after 1 hour exposure. In solution the lethal concentration of ethyl acetate was 1:100.In sugarcane medium, ethyl acetate could be detected after 24-hour incubation. More ethyl acetate was produced in young than in old sugarcane tissue. Both fungi grew better at 28 °C but produced more ethyl acetate at 20 °C, possibly explaining why pineapple disease was serious at the lower temperature. Ethyl acetate also could be isolated from soil containing diseased plant material.