Growth of Xanthomonas campestris pv. campestris populations at constant and variable temperatures

Quantitative data were collected to describe the relation between temperature and growth of the cabbage black rot pathogen,Xanthomonas campestris pv.campestris (Xcc). Relative growth rates derived from experiments at constant temperatures were used in dynamic simulation of bacterial population development. The relative growth rates were adequate to simulate growth ofXcc populations at constant temperatures but overestimated growth of populations at variable temperatures. This finding gives rise to the hypothesis, that under field conditions, disease development is slower than is expected on the basis of growth parameters obtained from studies with constant temperatures.     

Shaping a focus: wind and stochasticity

The diffusion theory of focus development permits development of a class of related models of different complexity. The basis of the present study is an earlier model which simulates a dual spore dispersal mechanism, consisting of a short-distance and a long-distance mechanism. This model was further elaborated by adding stochasticity and an external directional force simulating wind. Stochasticity was simulated through randomization of lesions produced by spores dispersed by the long-distance mechanism. The model was run twelve times for three values of spore partitioning over the two dispersal mechanisms combined with four values of wind speed. Model responses were sampled at three simulation time values: 40, 90, and 100 days. A major model response was the shape of a single focus, studied through snap-shot pictures of disease severity over the field. Real life phenomena, non-circular mother or central focus with ragged front and daughter foci, were reproduced. Throughout the simulations, maximum disease severity remained at the location of the initial inoculation. However, after all available sites around the centre were exhausted, the geometrical centre of the focus was apparently moved downwind. Number of daughter foci and their relative size were strongly dependent on the spore partitioning over the two dispersal mechanisms.     

The significance of VA-Mycorrhiza to future arable farming in the Netherlands

Present conditions of high inputs and narrow rotations render the importance of vesicular-arbuscular mycorrhiza (VAM) in arable farming in the Netherlands rather low. Changes expected in the near future under the influence of economic pressures and environmental problems may change this situation. This is discussed in the light of some such expected changes in Dutch arable farming.De huidige Nederlandse akkerbouw is zeer intensief. Ze heeft in het algemeen een nauwe rotatie met slechts enkele gewassen (aardappelen, granen en suikerbieten) en een ruime toepassing van kunstmest en bestrijdingsmiddelen. Het is te verwachten dat er onder invloed van toenemende problemen met het milieu, afnemende bodemproduktiviteit en lagere prijzen voor nu geteelde gewassen ten gevolge van overschotten een tendens zal zijn in de richting van een meer geïntegreerde landbouw. In de bestaande akkerbouw werken zowel hoge kunstmestgiften (met name N en P) en bestrijdingsmiddelen negatief op VAM-vorming. Naarmate het gebruik van kunstmest en bestrijdingsmiddelen in de nabije toekomst wellicht zal verminderen, kan worden verwacht dat zowel de aanwezigheid als het belang van VAM in de nederlandse landbouw zal toenemen. Het laatste zal vooral het geval zijn indien de vruchtwisseling wordt verruimd met de teelt van andere gewassen (zoals vlinderbloemigen) met een hogere VAM-afhankelijkheid dan die van het huidige gewasassortiment. Het zou zeer verstandig zijn zich reeds nu te beraden over vormen van bedrijfsvoering die VAM zoveel mogelijk stimuleren en daardoor meer profijt trekken van de door deze symbiose geboden voordelen.     

Phenotypic Characteristics of Xanthomonas campestris pv. campestris from Nepal

Twenty strains of Xanthomonas campestris pv. campestris (Xcc) were isolated from two major crucifer-growing valleys, Chitwan and Kathmandu in Nepal and characterized by biochemical and pathogenicity tests. Strains were homogeneous in bacteriological characteristics. The ability of a strain to induce high or low disease severity index (DSI) on three host plants, broccoli, cabbage, and cauliflower, was interpreted as virulence. Strains that were associated with high or low virulence were significantly different (P>0.05). No relationship between virulence and biochemical characteristics was observed.     

Systems approaches in epidemiology and plant disease management

This contribution to Zadoks’ liber amicorum reviews the developments in quantitative epidemiology during the last decades. It elucidates the progress in this field and shows how empirical crop protection with many phenomenological aspects transformed into a science-based (inter) discipline. The availability of experimental tools and the rapid development and introduction of computers enabled the application of systems approaches which stimulated a revolution in thinking and caused a considerable improvement of strategic and tactical decision making in crop protection. Zadoks played a crucial role in that development.     

Survival of Xanthomonas campestris pv. campestris in the phyllosphere and rhizosphere of weeds

The phyllosphere and rhizosphere of weeds are important niches for phytobacterial survival. The absence of information in Brazil regarding Xanthomonas campestris pv. campestris (Xcc), the causal agent of black rot in crucifers, motivated this study. Twenty‐six weed species belonging to 14 botanical families were included in field experiments between August 2014 and October 2015. Lepidium virginicum and Raphanus raphanistrum (Brassicaceae) demonstrated great potential for survival of Xcc in the phyllosphere, with the bacterium isolated after 56 and 70 days, respectively. Low variation between maximum and minimum temperatures, high rainfall and high relative humidity at specific times of the year contributed to longer Xcc survival periods in the phyllosphere of some species. Xcc survived in the rhizosphere only in R. raphanistrum, where it was isolated for up to 28 days. No relation was found between climatic factors and survival in the rhizosphere. The data indicate that control of brassicaceous weeds will contribute to the control of black rot.     

Genetic mapping and utilization of quantitative trichome-mediated insect resistance in potato

Introgression of trichome-mediated insect resistance from the wild speciesSolanum berthaultii has become a major focus of the potato improvement program at Cornell University during the past twelve years. Several quantitative characters are involved in this resistance which is effective against a wide range of pest types. Correlative biochemical assays have been developed to assay specific components of the resistance, and the effects of the resistance on the target pests have been studied. Quantitative laboratory assays and specific measurements of insect behavior and biology have increased the precision of selection and enable the investigation of the genetic control of the resistance.We are currently using restriction fragment length polymorphisms (RFLPs) for genetic mapping of factors controlling the trichome traits fromS. berthaultii. Backcrosses to both the wild and the cultivated species parents have been evaluated for phenotypes contributing to the resistance mechanism, including trichome density, sucrose ester and polyphenol oxidase production by the trichomes, and the enzymatic browning reaction responsible for insect entrapment. Genetic maps are being developed for these progenies, using RFLP markers previously mapped in potato. Field and greenhouse trials under insect infestations are also being conducted with the mapping progeny. Our goal is to locate genes responsible for quantitative insect resistance by correlating RFLP variation at mapped loci with the trichome phenotypes and insect resistance. Genetic markers for these traits will be useful in transfer of the effective wild chromosomal segments into and among tetraploid potatoes, and for a better understanding of the resistance mechanism.     

Effect of interrupted leaf wetness periods on pustule development of Puccinia recondita f. sp. tritici on wheat

In growth chamber experiments, seedlings of a susceptible wheat cultivar (Rubis) were inoculated with urediospores of wheat leaf rust. Inoculated seedlings were incubated in a moist chamber. The period of exposure to leaf wetness varied in duration and progressed with or without one or more interruptions of variable duration. During the interruptions the leaves with germlings were dry. At near-optimal temperature, leaf wetness periods of six h resulted in 60 to 65% of the pustules produced with 12 or 24 h wetness periods. Interruption of a 6 h leaf wetness period by a 1 h dry period was most damaging to the rust germlings at about three to four h after inoculation. The damage became visible as a reduction in the number of resulting pustules and as a prolongation of the median latency period. Epidemiological implications of leaf wetness interruptions are discussed.Kiemplanten van het vatbare tarweras Rubis werden geïnoculeerd met uredosporen van de bruine roest van tarwe. De proeven vonden plaats in klimaatkamers. Geïnoculeerde planten werden geïncubeerd in incubatiehokjes met een zeer hoge luchtvochtigheid, teneinde condensatiedruppels op de bladeren en aldus bladnatperioden te krijgen. De toegediende bladnatperioden varieerden in tijdsduur en zij verliepen met of zonder één of meer onderbrekingen, ook weer van variërende duur. Tijdens de onderbrekingen stonden de planten met roestkiemlingen in het licht; het blad was dan droog. Bij ongeveer optimale temperatuur en bladnatperioden van 6 uur was het aantal verkregen puistjes 60 à 65% van het aantal verkregen puistjes na een bladnatperiode van 12 of 24 uur. Onderbreking van de 6-urige bladnatperiode door een 1-urige droge periode was voor de roestkiemlingen het schadelijkst ongeveer 3 tot 4 uur na inoculatie. De schade kwam tot uitdrukking in het kleinere aantal geproduceerde puistjes en in een verlenging van de mediane latentieperiode. De epidemiologische betekenis van onderbrekingen van de bladnatperiode wordt besproken.     

Molecular aspects of the potato — Phytophthora infestans interaction

The fungal pathogenPhytophthora infestans is the causal organism of late blight, one of the most devastating diseases of potato. In the past, various aspects of the potato-P. infestans interaction have been studied extensively. In this paper we briefly review the current knowledge of the molecular events associated with the interaction and in addition we discuss a new approach for analyzing the molecular basis of pathogenicity ofP. infestans.     

Modelling the spread of Xanthomonas campestris pv. campestris in module-raised brassica transplants

A series of experiments was performed to quantify the rate of dispersal of Xanthomonas campestris pv. campestris in module-raised brassica transplants, in a simulated commercial plant-raising system. Seeds were sown in '308' module seed trays and set out in blocks in the glasshouse. Primary inoculum was introduced as inoculated seeds sown in one or more cells. Trays were watered via an overhead-gantry irrigation system, hand-watered or capillary-watered. Disease symptoms were monitored visually and the presence of the pathogen on samples of plants was monitored by leaf washing, dilution and plating on selective medium. Spread of symptoms was greatest in the gantry-watered trays, was very limited in hand-watered trays and was almost non-existent in capillary-watered trays. Dispersal of bacteria followed a similar pattern, but the proportion of plants contaminated was much greater than the proportion showing symptoms, and approached 100% after six weeks in the gantry-watered trays within 50 plants distance from a single primary infector. Models relating the proportion of plants with symptoms, or contaminated, to the distance from primary infector and time since sowing were fitted to the data. Predictions of the proportions of plants contaminated in commercial-scale blocks of transplants suggested that high levels of disease in the field could be explained by rapid rates of pathogen spread during plant-raising, and that the widely-used tolerance standard for seed health testing (0·01%) should be revised to 0·004%. In addition to seed health testing, control should focus on raising transplants under conditions that minimise the rates of disease spread and pathogen dispersal.     

Symptomless Spread of Blight-inducing Strains of Xanthomonas campestris pv. campestris on Cabbage Seedlings in Misted Seedbeds

Xanthomonas campestris pv. campestris induces two types of symptoms, namely, black rot and blight. Black rot symptoms are V-shaped lesions and black veins on the leaf, and blight symptoms are sudden collapse of interveinal tissues following the lack of veinal necrosis at early stages of infection. These two symptoms can occur simultaneously. However, the tendency to induce either symptom type is strain-dependent. Six strains were evaluated for their rate and pattern of spread in misted seedbeds by using strain-specific monoclonal antibodies and miniplate enrichment/ELISA. Data on pathogen incidence was defined as the presence of the pathogen in or on plants rather than visual symptoms. The results indicated that blight-inducing strains spread to more seedlings than black rot-inducing strains. The high incidences of blight-inducing strains in experimental plots were associated with non-randomness of spatial pattern of pathogen spread, indicating that high incidence is primarily due to the spread from adjacent plants by leaf contact and water splash. Most ELISA-positive seedlings were symptomless, indicating that the sensitivity of the system used in this study was adequate for detection of latent or epiphytic spread.     

Towards the isolation of resistance genes by transposon targeting in potato

A general strategy for the isolation of disease resistance genes is presented, employing a two-step approach of transposon targeting near genes of interest followed by transposon tagging. A library of transposon (Ac/Ds) transformants in a self fertile potato diploid are being mapped by deriving genomic DNA probes flanking the transposon containing T-DNA insertions with the inverse polymerase chain reaction and using these probes for RFLP analysis. We have produced a large number of transposon (Ac/Ds) transformants in a self fertile potato diploid. Genomic DNA probes, flanking the transposon containing T-DNA insertions, are produced by the inverse polymerase chain reaction (IPCR) and mapped by restriction fragment length polymorphism (RFLP) analysis in a segragating potato location. A transposon mapped close to a resistance gene can be recombined cis to the gene and used for efficient transposon targeting due to preferential transposition to linked sites.     

Specificity of polycllonal and monoclonal antibodies for the identification of Xanthomonas campestris pv. campestris

Polyclonal and monoclonal antibodies (PCAs and MCAs), produced to whole cells and flagellar extracts ofXanthomonas campestris pv.campestris (Xcc), respectively, were tested for specificity. In immunofluorescence microscopy (IF) the three PCAs tested, reacted at low dilutions with all Xcc strains, some other xanthomonads and non-xanthomonads. At higher dilutions most cross-reactivity with non-xanthomonad strains disappeared. However, the cross-reactivity with strains ofX. c. pv.vesicatoria (Xcv),X. c. pv.amoraciae (Xca) andX. c. pv.phaseoli var. fuscans (Xcpf) remained.Six MCA-producing cell clones viz. 20H6, 2F4, 18G12, 10C5, 17C12 and 16B5 were selected for specificity tests with an enzyme immunoassay (EIA), IF and a dot-blot immunoassay (DBI). None of the MCAs reacted with all Xcc strains in IF and EIA. In DBI, only MCAs 17C12 and 16B5 reacted with all Xcc strains. All six MCAs tested, cross-reacted in one of either tests with other pathovars ofX. campestris, such as Xcv or Xca. The MCAs were also tested in immunoblotting experiments using total bacterial extracts, cell envelope and flagellar extracts. MCAs 20H6, 2F4, 18G12 and 10C5 reacted with the lipopolysaccharide (LPS) of Xcc. MCAs 16B5 and 17C12 reacted with a 39 kilodalton and a 29 kilodalton protein, respectively.It is concluded that the PCAs and MCAs discussed in this study may be used for routine identification and differentiation of (a group of) Xcc strains. The significance of the cross-reactions with other pathovars ofX. campestris needs to be determined by testing seed lots.     

Prospects for improving potato crop protection against aphid vectors

Conventional ways to control aphid vectors on potato crops rely essentially on cultural and chemical methods. The extensive use of insecticides, too often applied regardless of real needs and consequences, has resulted in more and more failures in both controlling aphids and virus spread. This paper stresses the need to introduce more rationale in the operation of existing methods by improving the efficiency of some of them like forecasting systems, to assess the potential of others like fertilization and semiochemicals and to put most efforts on resistant cultivars so that integrated pest management can be operated in the near future.     

Optimal cultivar mixtures in focal plant diseases

This paper deals with a study on the effects of mixtures of partially resistant cultivars in focus forming plant diseases. The focus expansion velocity in a mixture of two partially resistant cultivars depends on four parameters, the focus expansion velocity in a crop of cultivar 1 only and in a crop of cultivar 2 only, the net-reproduction in a crop of cultivar 1 and of cultivar 2, respectively. An explicit equation is developed for the damage of a cultivar mixture where the crop is infected by two physiological races of the pathogen. It is shown that cultivar mixtures exist where damage is smaller than in crops of either of the two cultivars only. A simple explicit formula is given on the basis of which it can be decided whether an optimal mixture exists for a given pathosystem.     

Effect of culture filtrates of seventeen fungicolous fungi on sporulation of cucumber powdery mildew

Culture filtrates of 17 different fungal species thriving upon other fungi were tested for their ability to reduce sporulation of cucumber powdery mildew,Sphaerotheca fuliginea.All culture filtrates reduced the number of healthy conidiophores. However, the differences in activity between the various treatments were not as conspicuous as after application of spore suspensions. The best results were obtained with culture filtrates ofCalcarisporium arbuscula. These reduced the number of healthy conidiophores to ca. 2% of the unsprayed control plants.     

Effects of imazalil on sterol composition of sensitive and DMI-resistant isolates of Penicillium italicum

Imazalil differentially inhibited dry weight increase of 10-hour-old germlings of wild-type and DMI-resistant isolates ofPenicillium italicum in liquid malt cultures. EC₅₀ values ranged from 0.005 to 0.27 g ml^–1. In all isolates ergosterol constituted the major sterol (over 95% of total sterols) in the absence of the fungicide. Therefore, DMI-resistance cannot be associated to a deficiency of the C-14 demethylation enzyme in the ergosterol biosynthetic pathway. Imazalil treatment at concentrations around EC₅₀ values for inhibition of mycelial growth resulted in a decrease in ergosterol content and a simultaneous increase in 24-methylene-24,25-dihydrolanosterol content in all isolates. A correlation existed between the imazalil concentration necessary to induce such changes in sterol composition and the EC₅₀ values for inhibition of mycelial growth of the different isolates. The reason for the differential effects of imazalil on sterol composition in the variousP. italicum isolates may be due to decreased accumulation of the fungicide in the mycelium and to other yet non-identified mechanisms of resistance.Imazalil remt differentieel de toename in drooggewicht van 10-uur-oude gekiemde sporen van wild-type en DMI-resistente isolaten vanPenicillium italicum in vloeistofcultures van moutextract. De EC₅₀ waarden voor groei van de verschillende isolaten lopen uiteen van 0,005 tot 0,27 g ml^–1. In afwezigheid van het fungicide is in alle isolaten ergosterol het belangrijkste sterol (meer dan 95% van het totaal). DMI-resistentie kan daarom niet in verband staan met deficiëntie van het C-14 demethyleringsenzym in de ergosterol biosynthese. Imazalilbehandeling van mycelium bij concentraties rond de EC₅₀ waarde voor groeiremming, resulteerde bij alle isolaten in een afname van het ergosterolgehalte en een gelijktijdige toename van het gehalte aan 24-methyleen-24,25-dihydrolanosterol. Er bestaat dus een nauwe correlatie tussen de imazalilconcentratie die noodzakelijk is om vergelijkbare veranderingen in sterolsamenstelling te induceren en de EC₅₀ waarde voor remming van myceliumgroei van de verschillende isolaten. De differentiële effecten van imazalil op de sterolsamenstelling van de verschillendeP. italicum isolaten kunnen worden veroorzaakt door verminderde accumulatie van het fungicide in het mycelium en door andere, nog niet geïdentificeerde resistentiemechanismen.     

‘Plantibodies’: a flexible approach to design resistance against pathogens

Engineering resistance against various diseases and pests is hampered by the lack of suitable genes. To overcome this problem we started a research program aimed at obtaining resistance by transfecting plants with genes encoding monoclonal antibodies against pathogen specific proteins. The idea is that monoclonal antibodies will inhibit the biological activity of molecules that are essential for the pathogenesis. Potato cyst nematodes are chosen as a model and it is thought that monoclonal antibodies are able to block the function of the saliva proteins of this parasite. These proteins are, among others, responsible for the induction of multinucleate transfer cells upon which the nematode feeds. It is well documented that the ability of antibodies to bind molecules is sufficient to inactivate the function of an antigen and in view of the potential of animals to synthesize antibodies to almost any molecular structure, this strategy should be feasible for a wide range of diseases and pests.Antibodies have several desirable features with regard to protein engineering. The antibody (IgG) is a Y-shaped molecule, in which the domains forming the tips of the arms bind to antigen and those forming the stem are responsible for triggering effector functions (Fc fragments) that eliminate the antigen from the animal. Domains carrying the antigen-binding loops (Fv and Fab fragments) can be used separately from the Fc fragments without loss of affinity. The antigen-binding domains can also be endowed with new properties by fusing them to toxins or enzymes. Antibody engineering is also facilitated by the Polymerase Chain Reaction (PCR). A systematic comparison of the nucleotide sequence of more than 100 antibodies revealed that not only the 3-ends, but also the 5-ends of the antibody genes are relatively conserved. We were able to design a small set of primers with restriction sites for forced cloning, which allowed the amplification of genes encoding antibodies specific for the saliva proteins ofGlobodera rostochiensis. Complete heavy and light chain genes as well as single chain Fv fragments (scFv), in which the variable parts of the light (V_L) and heavy chain (V_H) are linked by a peptide, will be transferred to potato plants. A major challenge will be to establish a correct expression of the antibody genes with regard to three dimensional folding, assembly and intracellular location.     

Differentiation of Xanthomonas campestris pv. Phaseoli from Xanthomonas campestris pv. Phaseoli var. Fuscans by PFGE and RFLP

Xanthomonas campestris pv. phaseoli and X. campestris pv. phaseoli var. fuscans, the causal agents of the common and fuscous bacterial blight of beans, appear to be phenotypically identical except that the latter can produce a melanin-like pigment in culture. Ten isolates of X. campestris pv. phaseoli and 12 isolates of X. campestris pv. phaseoli var. fuscans were examined using pulsed-field gel electrophoresis (PFGE) and restriction fragment length polymorphism (RFLP). The average genome sizes for X. campestris pv. phaseoli and X. campestris pv. phaseoli var. fuscans were 3850.6±48.9 and 3584.3±68.1kb respectively. The genetic relatedness of the isolates was determined from macrorestriction patterns generated using XbaI. Cluster analysis indicated that the non-fuscous and fuscous strains are distinct. RFLP results, based on the highly conserved hrp genes and a pectate lyase gene from Xanthomonas, also indicated that the two bacteria are genetically different. The results obtained in this study suggest that this pathovar can be segregated into two subgroups under a recently proposed reclassification of the Xanthomonas genus.     

Surveys of cereal diseases and pests in the Netherlands. 6. Occurrence of insect pests in winter wheat

Between 1980 and 1986, commercial winter wheat fields at the milky-ripe stage were surveyed for insect pests. Leaf injuring insects were common. On average 42% and 95% of the surveyed fields were injured by leaf miners and the cereal leaf beetle, respectively. On average 14% of the leaves was skeletonised by the cereal leaf beetle and leaf injury tended to be high in seasons after winters with low temperatures. Blossom midges were the most important midges. In the period 1980–1984, the orange and lemon blossom midge infested 71 and 21% of the surveyed fields, respectively, Insecticides were mainly used to control aphids. Despite control, on average 80% of the fields and 22% of the tillers were infested by aphids.