Biology, pathotype, and virulence of Globodera rostochiensis populations from Kenya.

Specific scopeThis Standard describes a national regulatory control system for Globodera pallida and Globodera rostochiensis.

Testing of potato varieties to assess resistance to <i>Globodera rostochiensis</i> and <i>Globodera pallida</i>

The potato cyst nematodes Globodera rostochiensis (Woll.) Skarbilovich and G. pallida (Stone) originate from the Andes region in South America and have been introduced into Western Europe since 1850. Both species are successful colonizers. Once primary founders have established vital populations, an area is rapidly colonized by secondary founding events. The mode of spread results in patchy distribution patterns. Analyses of the processes that influence the spatial variations in virulence are of major importance for the control by means of host plant resistance. The ability to unravel the mosaic distribution patterns of the two species and their pathotypes enables breeders and growers to anticipate on the dynamics of virulent populations. The aim of this thesis was to analyse the intra- and interspecific variation of G. rostochiensis and G. pallida in the Netherlands and to obtain insight in the processes that determine the spatial variations in protein, DNA and (a)virulence polymorphisms.interspecific variationThe molecular variation between the sibling species G.rostochiensis and G.pallida is remarkably large. The RAPD technique revealed a total of 250 DNA fragments, of which only nine DNA fragments were common to both species (Chapter 3). Similar results were obtained with the AFLP assay. A total of 1000 AFLP fragments was amplified, of which only 64 fragments could be identified in both species (Chapter 4). These results agreed with previous investigations using 2-DGE and show that morphologically nearly indistinguishable organisms can be quite distinct the molecular level.The extensive genetic differentiation of G.rostochiensis and G . pallida offers perspectives for the development of a diagnostic assay. In addition the PCR technique enables the amplification of species specific fragments from small numbers of cysts extracted from soil samples. For example, G. rostochiensis specific DNA fragments can be amplified from single juveniles (Chapter 1).intraspecific variationThe intraspecific variation of G.rostochiensis as revealed by RAPDs and AFLPs is relatively low (Chapter 3 and 4). The proportion of polymorphic DNA fragments among nine G. rostochiensis populations was 19% and 15.8%, respectively. Three clusters of populations were identified and each cluster could be described by one or more specific DNA fragments.The intraspecific variation of G.pallida, as revealed with RAPDs or AFLPs, is larger in comparison with G.rostochiensis (Chapter 3 and 4). The proportion of polymorphic RAPD fragments among 17 populations was 46%. For the AFLP assay this figure was 23% polymorphic AFLP fragments among 15 populations. The majority of the populations displayed a continuous range of variations. Only a few clearly diverged clusters could be discriminated on the basis of specific DNA fragments.Various groups have applied the RAPD technique to study the genetic divergence among conspecific populations of plant parasitic nematodes. The validity of this technique was evaluated in chapter 4 by comparison of the clustering of 36 G.pallida populations based on RAPD and 2-DGE data. Both data sets demonstrated that the majority of the G.pallida populations were not clearly differentiated from each other. The overall correlation between the distance matrices derived from both data sets was low. Careful examination of the separate dendrograms showed similarity in clustering only for clearly diverged populations or groups of populations.Although the AFLP and RAPD technique are simple, fast and require only minute amounts of biological material, they are not suitable to resolve the subtle differences among potato cyst nematode populations. Quantitative variation in allele frequencies is often not resolved with those techniques, which is, among others, due to the virtual inability to recognize co-dominant alleles and the non-linear amplification of DNA fragments. In chapter 6 and 7 the genetic variation was studied by analysing pools of individuals with 2-DGE. The ratios between the protein quantities produced by the codominant alleles are appropriate measures for the allele frequencies. The correctness of this method has been confirmed by 2-DGE of single individuals (De Boer etal ., 1992)bottleneck effects on the secondary foundersThe intraspecific variation among potato cyst nematode populations in Europe is predominantly determined by the genetic constitution of the primary founders, directly or indirectly introduced from South America, and the effect of random genetic drift on the secondary founders. To obtain insight in these processes, 226 G.pallida populations from the Netherlands were analyzed with 2- DGE. The results strongly suggest that these populations originate from one source, or in case of multiple introductions, from a number of sources with a similar genetic makeup.The genetic differentiation of the 226 G.pallida populations indicate that the colonization of the Netherlands has been accompanied by extensive random genetic drift. Only a limited proportion of the populations appeared to be identical. It is also shown, that the bottleneck effects differ between regions. Significant variation in population structure was observed between the three investigated areas. The genetic variation within G.pallida populations from north Friesland and the IJsselmeerpolders is in general smaller than within populations from the northeast Netherlands. A plausible explanation for this phenomena is the low multiplication rate of potato cyst nematodes due to wider crop rotation schemes in the IJsselmeerpolders and north Friesland. These factors result in a slow expansion of newly founded populations, which enhances the effect of drift.gene-pool similarity conceptThe gene-pool similarity concept rests on the hypothesis that in the absence of selection pressure by host plant resistance, degrees of similarity between populations revealed by molecular techniques are also reflected at virulence loci, including those not yet resolved. To test this concept, the genetic variation revealed by 2-DGE among 102 G.pallida populations was compared with the variation in virulence towards two resistant cultivars. This analysis showed that a decrease in genetic distance among populations is accompanied with a decrease in variation in virulence. In addition it is demonstrated that the gene pool similarity concept is also applicable to loci determining the variation in fitness among populations. The variation in Pf/Pi values among the 102 populations on Désirée is in general smaller between closely related populations than between distantly related populations.breeding for resistanceBreeding for resistance has been dominated by trial-and-error approaches, which has stimulated the view that control by means of host plant resistance is unavoidably a short-term approach due to the 'appearance' of virulent populations. The pervasive myth that breeding for resistance against potato cyst nematodes is a lost arms race is challenged by the results of this thesis. Until recently it was assumed that the genetic variation of G.pallida in the Netherlands was too large to produce potato cultivars with broad-spectrum resistance. In this thesis it is shown that the genetic diversity introduced from the Andes region has been limited and that the variation among the Dutch G.pallida populations is mainly the result of random genetic drift. The elaborate analysis of 226 Dutch G.pallida populations offers perspectives to obtain potato cultivars with broad and durable resistance. The gene pool similarities revealed by 2-DGE can be used as guidance in testing the effectiveness of new sources of resistance.

Potatoes are among the most profitable agricultural crops in arable farming in the Netherlands and consequently are grown as frequently as conditions allow. As a result Dutch farmers experienced huge problems with potato cyst nematodes during the last 50 years. In Chapter 1 an outline is presented of the situation of potato cyst nematode control in the 1980's, the problems, possible solutions and the research initiated, of which a part is presented in this thesis.In 1984 emphasis was put into research concerning the efficiency of soil fumigation on heavy marine clay soils where the majority of Dutch seed and consumption potatoes are grown. Both laboratory and field experiments were carried out to investigate the efficiency of 1,3 dichloropropene and metam-natrium. This required the processing of thousands of larvae suspensions. One of the most labourious and tedious occupations in nematological research is the counting of individuals of the pathogen.In Chapter 2 A GOP-302 image analysis system - Context Vision, Sweden - was used to automate the counting of large numbers of larvae-suspensions of Globodera rostochiensis and G. pallida . These suspensions originated from hatching tests, which were conducted to estimate percentage mortality in field and lab experiments of nematodes exposed to nematicides. The result is called ANECS ( A utomatic NE matode C ounting S ystem), a software program that can count up to 64 compartments with larvae suspensions successively without the aid of an operator. A special object carrier was developed. Images of up to eight object carriers (512 larvae suspensions) can be stored and image analysis can be suspended to off-office hours. The time needed to count one compartment was reduced by 80% to one minute compared to 'manual' labour while the time for probe preparation remained the same. The percentile error is highest at very low larvae densities (<20 per suspension) and is caused by pollution with small fibres carried by air during the handling of the larvae suspensions. This problem can be minimised by setting up clean-laboratory procedures. At least 95% of the larvae originating from hatching tests were recognized and counted. The program can and has been be adapted to count other nematode species or to suit more complicated problems like counting both larvae and eggs in one suspension.In Chapter 3 errors due to subsampling and laboratory procedures affecting the expected value and variance of cyst counts were investigated. Several fields, infested with potato cyst nematodes ( Globodera rostochiensis and G. pallida ) were sampled by collecting bulk samples of approximately 70 cores amounting to 1.8 or 2.5 kg soil from a number of square metre plots located in a regular grid pattern over a 0.33 ha area. Bulk samples from five fields, I-V, were thoroughly mixed and from one field, VI, lightly mixed, and subsequently divided into three subsamples of approximately equal weights. Two, sometimes three, subsamples were elutriated separately. Cysts were elutriated by two commercial laboratories, 1 and 2, and separated from the debris and counted at two research laboratories, 0 and 3.Random bulk samples from five fields, I-V, were divided into three portion after thoroughly mixing and taken to Laboratory 0, to compare elutriation precision and accuracy of commercial and scientific laboratories and to check the quality of mixing. To this purpose, pairs or triples were divided into classes. The expected value of the variance within pairs was estimated per class and could be described by a distribution function analogue to a negative binomial distribution, but with three in stead of two parameters. Cysts appeared to be randomly distributed in the well mixed samples, resulting in a binomial or trinomial distribution between pairs or triples. The expected values of the coefficient of variation associated with elutriation were 3.6, 9.6 and 5.5% in the Laboratories 0, 1 and 2, respectively. The upper 95% confidence limit,δ 0.95 , of coefficients of variation associated with elutriation in Laboratories 1 and 2, were estimated by the differences in 95% upper limits of coefficients of variation between the Laboratories 1 and 2 on the one hand and Laboratory 0 conversely. This difference,δ 0.95 , ranged from 73% to 42% for Laboratory 1 and from 43% to 19% for Laboratory 2 if 10 to 100 cysts were counted in samples. The consequences of these laboratory errors for the accuracy of sampling methods for both research and extension purposes are discussed.Nematicide trials require reliable results concerning the effect caused by the fumigant. The percentage mortality of potato cyst nematodes can be estimated by comparing the hatchability of untreated larvae with that of nematicide treated larvae. In Chapter 4 , research is described to improve the quality of hatching tests. Hatching tests using potato root diffusate are labourious and yield quite variable results. Sources of variability were identified and analysed, and solutions were presented. A method was developed to conduct hatching tests using inert materials so that the total variation at the end of the test is minimized.A number of hatching tests was carried out to increase reliability, optimize the method and limit the amount of work. Thus, it was possible to obtain a coefficient of variation ( cv ) of the hatching process which is in accordance with the combined errors expected when a certain number of cysts is treated and eggs are used in a hatching test. An Appendix is provided listing the different errors and ways to calculate and cope with them.The results indicate that the hatching process is no longer an important source of variation for the end result. All variation higher than expected could be explained by variation between replications of batches with the same treatment, indicating that small differences in nematicide application cause major differences in the end result. The treatment effect was more important in field experiments than in laboratory experiments.The hatching curve could be described adequately by a log-logistic curve with 3 parameters (λfinal number of hatched larvae,αtime,βslope parameter). Addition of a fourth parameter (γ, incubation time) improved the fit of the hatching curve significantly. Using the log-logistic model, final hatch can be predicted with a certain error before the actual hatching test ended, but in general final hatch is underestimated. When an error of 5% is accepted, the length of time required to perform a hatching test of a laboratory experiment can be reduced by 80% for untreated batches and by 40 to 80% for batches treated with nematicides. Acceptable reduction is negatively correlated with the concentration of the fumigant used.Hatching tests with cysts originating from field experiments are unsuitable for prediction using a time limited data set. In cyst batches from the field compound hatching curves could be distinguished in 4 out of 6 fields, indicating that the soil samples contained at least two fractions of cysts with different hatching responses. Prediction would cause a significant underestimation of final hatch and consequently an overestimation of mortality.Because of its high vapour pressure, 1,3-dichloropropene is primarily used on marine clay soils. In Chapter 5 a laboratory experiment is described investigating the two stereo-isomeres of 1,3-dichloropropene for their efficiency in killing nematodes. Batches of increasing numbers of Globodera rostochiensis cysts were exposed to a range of concentrations of the (E)- and (Z)-isomers of 1,3-dichloropropene. The cysts were of identical origin. Temperature during treatment was 10 oC, humidity 100% and time of exposure 8 days. The integrals of concentration time products ( CT ) created were 0, 3, 7, 14, 31, 60, 125, 242, and 437μg/ml·day for the (E)-isomer and 0, 3, 16, 59, 240, and 419μg/ml·day for the (Z)-isomer. Survival was estimated with hatching tests 1.5, 3, and 7 months after treatment.The relationship between dosage of (E)-isomer and numbers of hatchable nematodes followed a log-logistic equation at all hatching dates. Hatchability, and therefore lethal dosages, increased as hatching tests were more delayed. Seven months after treatment, practically all treated nematodes had recovered and hatchability of treated and untreated nematodes was the same. A log-logistic relationship was also found for dosage (Z)-isomer and numbers of hatchable nematodes 1.5 month after treatment. When hatching tests of nematodes treated with the (Z)-isomer were delayed till 3 and 7 months after treatment, the results were better explained by a compound model, assuming two independent log-logistic effects, one stimulating hatch at low dosages and one reducing hatch at all dosages. Only the (Z)-isomer of 1,3-dichloropropene was effective as a nematicide.Chapter 6 presents research concerning the efficiency of standard doses of 1,3 dichloropropene in fields with a high silt content. Three fields of marine clay soil were fumigated with 150 l/ha 1,3-dichloropropene (DD) (Teleone II TM, Shell 95 TM). On three dates after application, concentrations of Z- and E- 1,3-dichloropropene were measured per 5 cm layer of soil to a depth of 40 cm and integrals of concentration time products were calculated. When the fumigant was no longer detectable, a top soil treatment with either 150 l/ha metam-sodium or 180 kg/ha dazomet (active compound methyl isothiocyanate) was applied, followed immediately by autumn ploughing. Soil samples were taken before and after fumigation and after the top soil treatment to extract potato cyst nematodes (PCN). Survival was determined by means of hatching tests. Mortalities after the DD treatment, defined as 100 - % survival, were estimated per 5 cm layer of soil to a depth of 30 cm to construct dosage response curves. Fumigation with DD killed 48, 48 and 72% of the PCN per field, respectively. Accelerated breakdown of DD by microorganisms accounted for the two lower mortality rates.The additional top soil treatment with metam-sodium increased mortality to 90% or more. Dazomet, however, was less effective (53 and 80%) considering that twice as much of the active compound was applied as in the metam-sodium treatment. Multiplication of hatched larvae originating from the injection layer after the DD treatment was 25% less than that from untreated plots. This was caused by a lower fraction of larvae developing into cysts. PCN could be retrieved from soil layers as deep as 80 cm below the surface. Fumigation reached only a fraction of the infested soil, down to 25-30 cm. The infestation foci were so small compared to the standard minimum area fumigated (1 ha) that 90% of the active compound would be wasted on non-infested soil. Soil fumigation, whether or not combined with an additional top soil treatment, will seldom be profitable. Monitoring for infestation foci is recommended.As soil fumigation was not a viable option to keep potato cyst nematodes in check on heavy marine clay soils, another way of control had to be found. Research was focussed at the development of sampling methods for the detection of small infestation foci with high reliability (≥90% probability of detection). Precautionary soil fumigation can be avoided, the area where a control measure has to be applied can be minimized to the actual infestation, and detection occurs so early that (partially) resistant potato cultivars can be grown without significant yield reduction as population densities are still low.Research in the Flevopolders yielded promising results. Therefore, in 1990, a research program was initiated to develop new sampling methods for the detection of patchy infestations of potato cyst nematodes ( Globodera rostochiensis and G. pallida ) with known accuracy in all potato cropping areas of The Netherlands. Patchy infestations in cropping areas of the provinces of Zeeland, Friesland, Groningen and Drente were sampled to validate a model based on data from cropping areas in Flevoland and to determine whether one detection method could meet the requirements of all cropping areas in The Netherlands. The results are presented in Chapter 7 . Eighty two fields were presampled to locate patchy infestations using a coarse sampling grid (8 · 3 m). Parts of thirty seven fields, containing one or more foci, were sampled intensively by extracting at least 1.5 kg of soil per square metre (1.33 · 0.75 m). Forty foci were analysed for spatial distribution characteristics of cysts using Generalized Linear Models (GLM's) and classical Multiple Linear Regression Analysis, differing in assumptions about the distribution of the input variable (number of cysts per kg of soil).The results showed that the data from all investigated cropping areas fit well to an exponential model with two parameters, the length and width gradient parameters. Significant differences in these parameter values between cropping areas could not be demonstrated. As both parameters follow a normal distribution, the probability of any combination of these parameters can be described by a bivariate normal distribution. Gradient parameters were correlated but significant correlations between these parameters and certain variables, such as the nematode species involved ( G. pallida or G. rostochiensis ), the time interval between sampling and the last potato crop, soil type, cropping frequency and cyst density in the focus centre could not be demonstrated. It can be concluded that one detection method for small infestation foci suffices for all investigated cropping areas. Its expected accuracy is independent of soil type, potato cyst nematode species, cropping frequency or time interval between sampling and last potato crop.In Chapter 8 the model for infestation foci developed in the previous chapter was applied for practical usage. A computer program called SAMPLE was developed to evaluate existing and create new sampling methods for the detection of patchy infestations or 'foci' of the potato cyst nematode ( Globodera spp.). By combining a model for the medium scale distribution of cysts, which provides the expected population densities at each position within the focus, and a model for the small scale distribution within square metres (negative binomial distribution) SAMPLE allows to simulate sampling procedures.The importance of the parameters of the two distribution models - the length and width gradient parameters for the medium scale distribution and the aggregation factor k of the negative binomial distribution for the small scale distribution - was investigated by sensitivity analyses. The aggregation factor k proved to be less important when calculating the average detection probability of a focus than the length and width gradient parameters. Several existing versions of the statutory sampling method used in The Netherlands were tested for their performance on a standard infestation focus with a central population density of 50 cysts/kg soil.The standard focus is small enough to use resistant potato varieties as a control measure without noticeable yield reductions in a 1:3 potato crop rotation. As the statutory soil sampling methods did not perform with the desired average detection probability, set at 90%, the program was used to develop several new sampling methods for focus detection and to investigate their performance. SAMPLE is a tool to develop sampling methods on demand for every possible combination of characteristics required for use by seed and ware potato growers (recommendations for optimum control measures leading to maximum returns, Integrated Pest Management) and by governments (legislation, quarantine and export protection).For advisory purposes a model is required describing the relation between the number of potato cyst nematodes and tuber yield. A stochastic model with biologically relevant parameters was available. In Chapter 9 the direct relation between the number of potato cyst nematodes and plant growth is described and used to deduce the relation between nematode density and yield reduction of total plant weight and tuber yield. The relation between small and medium initial population densities and the relative total plant weight was derived as cross sections at right angles to the time axis of a growth model with three dimensions: time after planting t , relative total plant weight Y and relative growth rate r p /r 0 . The relative growth rate is the (constant) ratio between the growth rate r p of plants of a certain weight at a nematode density P and the growth rate r 0 of (younger) plants of the same weight without nematodes. Therefore, the ratio between the time after planting that plants need to reach a certain weight in the absence of nematodes and at nematode density P, t 0 /t p equals the ratio r p /r o (2).The relative growth rate r p /r o = k + (1 -k )0.95 P/T -1for P > T and = 1 for P ≤T (3). Formally, k is the minimum relative growth rate as P →∞. As a result the arbitrary equation y = m + (1- m )0.95 P/T- 1for P > T and = 1 for PT (6) also applies to the relation between small and medium initial population densities and relative total plant weight. T is the tolerance limit, below which growth and yield are not reduced by nematodes; m is the relative minimum yield.The relations between small and medium initial population densities of potato cyst nematodes and relative tuber weight of potatoes can be derived from the growth model in an analogous way. However, there is one complication: tuber initiation does not start at the same haulm weight in plants with and without nematodes, but at the smaller haulm weight the larger the nematode density. As a consequence, tuber weights of plants with a certain total weight at nematode density P are not equal to those of plants with the same total weight without nematodes, but r p Δ t units of weight larger,Δ t being the difference between the actual time of tuber initiation and the time total plant weight becomes the same as that of plants without nematodes at the initiation of tuber formation.Relative total and tuber weights of plants with 'early senescence' and at large nematode densities are smaller than estimated by the model and equation (2). This indicates that at large initial population densities growth reducing mechanism(s) become active that were not operating at smaller densities.In Chapter 10 an advisory system is presented for the management of potato cyst nematodes ( Globodera pallida) . It emphasizes the use of partially resistant potato cultivars, which provide the possibility of keeping population densities of potato cyst nematodes at a low level in short fixed rotations. Using stochastic models based on the population dynamics of potato cyst nematodes and the relation between pre-plant nematode densities and relative yield it is possible to calculate the probabilities of population development and the reductions in yield caused by these population densities. A simulation model is developed which integrates both models, using the frequency distributions of some of the most variable parameters relevant to a particular combination of potato cultivar and nematode population. Also, the natural decline in population density when non-hosts are grown is incorporated in the model.The model makes it possible to calculate the probability of a certain yield reduction, given a certain potato cultivar, nematode population and rotation. Therefore, it becomes feasible for a farmer to evaluate risks and the costs of different control measures in fixed rotations. The application of this model in the starch potato growing areas could lead to significant improvements in financial returns and a major reduction of the use of nematicides.In Chapter 11 we describe the 'Seinhorst research program' initiated by Dr J.W. Seinhorst, former head of the Nematology Department of the IPO-DLO. It consists of an empiric philosophy, the scientific methods applied, and the models developed at the IPO-DLO during the last 45 years of nematological research, including the 13 years in which the research described in this thesis was carried out. All theories of the Seinhorst research program are developed by searching for recurring regularities (patterns) in a collection of observations, named 'the empirical base'. To prevent " ghost theories from sloppy data " all assumptions underlying the empirical base are carefully described in theories with respect to methodology and technology, including statistics.The patterns to be recognized are summarized by mathematical equations, which must be connected with biological processes to bridge the gap between 'normal' language and mathematical language for the description of biological theories. Often, the patterns result from more than one biological process. If so, the basic patterns are disentangled from one another using a method of pattern analysis. The procedure is best carried out when only a limited number of more or less congruent patterns are involved. Therefore, attention must be given to the choice of the hierarchic level and the complexity of the investigated system. Investigations proceed from simple experimental systems to complex natural systems at a hierarchic level that is neither so high that manifesting processes are very dissimilar nor so low that one runs the risk of describing processes irrelevant for the purpose of the investigation.In the 'Seinhorst Research Program' this purpose is finding methods for improvement of financial returns of host crops attacked by plant-parasitic nematodes through calculating risks of nematode population development and subsequent yield reduction. Pattern analysis yields theories about causes of phenomena observed at the investigated hierarchic level and about properties of processes at the nearest lower hierarchic level. Predictions at the next higher hierarchic level are made by synthesizing several patterns in (stochastic) simulation models. Synthesis is also applied to compound patterns of processes in simple experimental systems, with the objective to explain complicated patterns in complex systems.In the Conclusions ( Chapter 12 ) an overview is presented of the practical results and aspects of the research effort described in this thesis. Some comments are made on the present state of affairs concerning potato cyst nematode control in the Dutch seed- and ware potato growing areas.

Potato cyst (Globodera spp.), root-knot (Meloidogyne spp.), and root lesion (Pratylenchus spp.) nematodes are the most economically important nematodes of potatoes in temperate climates. Potato cyst nematodes occur worldwide and include two species, Globodera pallida (the white cyst nematode) and Globodera rostochiensis (the golden nematode), each composed of several pathotypes. The gene H1 which confers a high level of resistance to the golden nematode pathotype Rol, has been bred into several potato cultivars that are grown worldwide. Mapping of the H) gene has led to the development of a molecular marker to screen segregating populations for resistance to the golden nematode and to monitor the use of resistant cultivars in statutory control programs. Pathotype Ro2 of the golden nematode that overcomes H1-mediated resistance was recently discovered in New York State in the United States, and a high level of resistance to this pathotype has been identified. Several sources of resistance to G. pallida are known and have been used in traditional potato breeding programs to develop cultivars resistant to this species. Molecular markers that are closely linked to genes for resistance to two G. pallida pathotypes have been identified in different regions of the potato genome. Although resistance to root-knot and root lesion nematodes has been identified, limited success has been realized in the use of this resistance either through classical or molecular techniques to manage these nematodes on potatoes. Engineered resistance genes are currently being developed that may prove effective against both species of potato cyst nematodes as well as other nematode parasites of potato.

Potato cyst nematodes (PCN), such as Globodera rostochiensis and Globodera pallida, are quarantine restricted pests of potato causing major yield and financial losses to farmers. G. rostochiensis was first reported from Kenya’s key potato growing area in 2015. We sought to determine the diversity, prevalence and distribution of PCN species across the country by conducting a country-wide survey between 2016 and 2018, which included a more focused, follow-up assessment in three key potato growing counties. A total of 1,348 soil samples were collected from 20 potato growing counties. Information regarding local potato farming practices, potato cultivar use, their diversity and availability was also recorded. PCN cysts were obtained from 968 samples (71.8%) in all the counties surveyed, with Nyandarua County recording the highest PCN field-incidence at 47.6%. The majority of PCN populations, 99.9%, were identified as G. rostochiensis, while G. pallida was recovered from just one field, in a mixed population with G. rostochiensis. Inconsistencies in PCR amplification efficiency was observed for G. rostochiensis using the recommended EPPO primers, compared with ITS primers AB28/TW81, indicating that this protocol cannot be entirely relied upon to effectively detect PCN. Egg density in Nyandarua County varied between 30.6 and 158.5 viable eggs/g soil, with an average egg viability of 78.9 ± 2.8% (min = 11.6%, max = 99.9%). The PCN-susceptible potato cultivar named Shangi was the most preferred and used by 65% of farmers due to its shorter dormancy and cooking time, while imported cultivars (Destiny, Jelly, Manitou, and Markies) with resistance to G. rostochiensis were used by 7.5% of farmers due to unavailability and/or limited access to seeds. Thus, most farmers preferred using their own farm-saved seeds as opposed to purchasing certified seeds. Establishing the distribution and prevalence of PCN and elucidating the local farming practices that could promote the spread of PCN is a necessary precursor to the implementation of any containment or management strategy in the country and ultimately across the region.

SUMMARYThe control of potato cyst nematode (Globodera rostochiensis) by the oxime‐carbamates aldicarb and oxamyl was tested in four fields in Scotland. Dazomet was tested in three of these fields and carbofuran in one. In untreated plots in the three most heavily infested fields Maris Piper (resistant) yielded better than Pentland Crown (non‐resistant). All nematicides increased the yields of both potato cultivars but had a greater effect on the yield of Pentland Crown. Dazomet increased yields of tubers most. Heavy nematode infestation reduced yield of tubers more in a sandy soil than in two sandy loams. In a field with few potato cyst nematodes nematicides did not significantly affect tuber yields.Although the nematicides greatly increased yields, they were not completely effective in controlling potato cyst nematodes. In treated plots in the lightly infested field, there were more nematode eggs following a crop of Pentland Crown than before. In contrast, Maris Piper markedly decreased post‐cropping populations and except at one site, where dazomet further decreased nematode numbers, combining nematicides with the resistant cultivar failed to decrease nematode numbers further. Nematicides decreased the numbers of larvae invading potato roots by up to 95%, oxamyl at 5–6 kg/ha being consistently the best treatment.

Several measures have been recommended in the control of potato cyst nematodes (PCN) with resistant potato cultivars being considered as the most practical and affordable for smallholder potato farmers in Kenya. However, the level of resistance in locally grown potato varieties is yet to be established. The aim of this study was to screen Kenyan potato cultivars against PCN under greenhouse conditions. Eleven potato cultivars namely Shangi, Dutch Robijn, Sherekea, Nyota, Roseline tana, Tigoni, Unica, Asante, Chulu, Kenya Mpya and Arka were screened with Desiree (susceptible variety), Manitou (resistant cultivar) as controls. For each potato cultivar, there were two sets of plants with the first set being inoculated with 50 cysts, while the second batch was nematode-free. The treatments were arranged in a completely randomized design with three replications. A scale of 1-9, with 9 indicating the highest level of resistance was used in the assessment of disease severity. Nematode infestation caused a reduction in root mass across the 11 cultivars from 20 to 100% compared to uninoculated controls. Reproductive index of PCN viable eggs across the 11 cultivars was &lt;1 compared to control (Desiree). Potato cultivars Shangi, Tigoni, Dutch, Chulu, Asante, Unica, Arka, Kenya Mpya, and Roseline Tana had a severity score of 1-3 (&gt;95%), hence were considered to be susceptible to PCN. The cultivars Sherekea and Nyota had a severity score of 4-6 (&lt;25%) and hence were considered partially resistant to PCN. The findings of this study provides a basis of integrating partially resistant potato cultivars into PCN management in smallholder farms. Keywords: Cyst viable eggs, reproductive index, resistance, susceptible, smallholder farmers, severity score.

Istifadah N, Pratama N, Taqwim S, Sunarto T. 2018. Effects of bacterial endophytes from potato roots and tubers on potato cyst nematode (Globodera rostochiensis). Biodiversitas 19: 47-51. Bacterial endophytes are bacteria that inhabit plant tissues without causing any diseases. The endophytes existence may have negative, neutral, or positive effects on the host plants. This paper discusses the effects of bacterial endophytes isolated from potato roots and tubers on potato growth and their abilities to suppress potato cyst nematode, Globodera rostochiensis. The bacterial endophytes were isolated from roots and tubers of potatoes obtained from six plantation areas in West Java. The endophyte isolates were examined for their effects on potato growth. The non-pathogenic isolates were tested for their abilities to suppress G. rostochiensis in vitro and in potato plants. The results showed that from 88 bacterial endophyte isolates obtained, 13 isolates caused rot in potato seed pieces, 22 isolates inhibited the potato growth, while, 2 isolates increased the growth, and as many as 51 isolates did not influence the growth. The in vitro test using the isolate culture filtrate revealed that there were seven isolates that caused mortality of G. rostochiensis juvenile-2 by 67.5-97.7%. These isolates, however, were not effective in damaging the nematode eggs. In the greenhouse experiment, the bacterial endophyte isolates suppressed the number of cysts by 51.7-65.4% and that of the juvenile-2 of G. rostochiensis by 48.6-76.4%.

Potato cyst nematodes (PCN) cause an overall 9% yield loss of total potato production worldwide. Research on sustainable management of PCN is still under progress. Two microbial fermentation products (MFPs) from Alltech, a proprietary blend formulated with a bacterial fermentation media and a Cu component (MFP5075), and a microbial based product (MFP3048), were evaluated against the PCN Globodera rostochiensis. In laboratory tests, effectiveness of the MFPs was recorded in terms of PCN juveniles (J2) hatching from cysts, J2 mortality and their attraction toward potato roots using pluronic gel. Greenhouse trials were conducted to study the effect of the products on PCN infestation in potato plants and a pilot scale experiment was conducted to study the impact of these MFPs on nematode biodiversity in garden soil. All treatments were performed within a concentration range of 0, 0.5, 1, and 2% (v/v) MFP5075 and 2, 6, 10, and 20 g/10 ml (w/v) MFP3048. The attraction assay, juvenile hatching and the PCN infestation in plants results were compared with those in an untreated control and a commercial nematicide (Nemguard™) treatment. After 24 h of treatment with 0.5 and 1% MFP5075, a 13-fold and 43-fold reduction, respectively, relative to J2 survival was recorded compared to that of untreated control. However, no J2 survived at 2% and above concentration of the MFP5075 treatment. Treatment with MFP3048 was effective in causing mortality of J2 only after 48-h. In the attraction assay, a 20-fold and 8-fold reduction in number of J2 attracted toward potato roots was observed, when treated with MFP5075, compared to the untreated and the Nemguard™ treatment, respectively. Subsequently, 30–35 PCN cysts were treated with both products dissolved in potato root diffusate and the results were recorded in terms of number of J2 hatched in each treatment after 10 days. No J2 hatched in the MFP5075 treatment, whereas mean numbers (±SE) of 243 ± 11.5, 30 ± 2.5, and 1.3 ± 0.6 J2 were noted in the untreated control, MFP3048, and the Nemguard™ treatment, respectively. The treatment with the MFPs compromised the integrity of the unhatched J2, which looked granular, whereas the internal organs of the unhatched J2 could be clearly identified in the untreated control. In plant infestation studies, treatment with MFP3048 and MFP5075 caused 90.6 and 84.9 percent reduction in PCN infestation, respectively, in terms of cysts developed on roots compared to untreated control. Overall, results indicate that the MFPs could potentially provide a promising alternative for sustainable PCN management.

The potato cyst nematodes (PCN) Globodera rostochiensis and Globodera pallida are the very important quarantine nematode pests of potato [Stone 1973]. Both species cause serious potato tuber yield losses. These species are subjected to strict quarantine regulations in many countries (EPPO 2017). G. rostochiensis was detected in Sichuan and Yunnan province, China in 2022 (Jiang et al. 2022). A survey for cyst nematodes in potato fields was conducted in Guizhou Province from 2018 to 2020. A total of 200 samples, including roots and soil, were collected from 40 potato fields in Hezhang (N27 06.145, E104 39.153) and Weining (N26 50.541, E104 09.885) counties in Guizhou Province, China. The Cobb decanting and sieving method was used to isolate cysts and J2s from the soil samples (Southey 1986). The potato roots were stained with acid fusion to observe cyst development. Morphological and molecular analyses indicated that 27 (13.5%) of the samples contained G. rostochiensis. The cyst density ranged between 1-85 cysts per 100 cm3 of soil and a mean density was 15 cysts per 100 cm3 soil. The smoothly rounded cysts were in brown and golden color, and the terminal cone was absent and circumfenestrate. The key morphometrics of cysts (n=20) were 695 ± 26 (685-757) μm in length excluding neck and 690 ± 30 (668 to769) μm in width; the number of cuticular ridges between anus and vulval fenestra was 16.3 ±2.1 (14 to 18); fenestral length was 15.1 ± 2.1 μm (13.18 to 19.27); distance from anus to the edge of fenestra was 61.12 ± 8.9 (49.22 to76.27) μm; and Granek's ratio was 4.54 ± 0.8 (3.97-5.26). The key morphometrics of J2 (n = 20): 468.0 ± 20.1 (427 to - 521) μm in body length, 20.58 ± 0.7 (20.2 to 21.8) μm in stylet length, 43.9 ± 5.6 (40.3 to 53.9) μm in tail length, and 23.1 ± 1.8 (21.77 to 25.32) μm in hyaline region length. The cyst and J2 morphologies were consistent with those of G. rostochiensis (Subbotin et al. 2010, EPPO 2017). Genomic DNA was isolated from cysts (n=20). DNA extraction was performed in a volume of 20 μl containing 3 μl 10× PCR buffer, 3 μl Proteinase K (600 μg μl-1), 14 μl distilled water and a single cyst was added and ground in an ice bath as described by Ou et al. (2008). The internal transcribed spacer (ITS) regions were amplified using the universal primers: rDNA1 (5'-TTGATTACGTCCCTGCCCTTT-3') and rDNA2 (5'-TTTCACTCGCCGTTACTAAGG-3') (Fleming 1998 ), and the 28S rDNA-D2/D3 regions were amplified using the primers: D2A (5´-ACAAGTACCGTGAGGGAAAGTTG-3´) and D3B (5´-TCGGAAGGAACCAGCTACTA-3´) (Subbotin et al. 2006). After the brackets at the beginning and end of the sequences were closed-up, the ITS rDNA sequences (GenBank Accession No. MZ042367 and MZ042368) showed 99.66% - 99.92% identity to G. rostochiensis sequences available in GenBank (FJ212166.1, GQ294513, FJ212164.1 and KJ409617.1). Sequences from the 28S region (GenBank Accession No. MZ057597 and MZ057598) were 99.23% - 99.74% similar to those of G. rostochiensis isolate from Slovakia (KJ409625.1), Italy (KJ409631.1) and United Kingdom (KJ409633.1). We used species specific primers ITS5（5'-GGAAGTAAAAGTCGTAACAAGG-3'）and PITSr3 (5'AGCGCAGACATGCCGCAA-3') to amplify the product (Bulman & Marshall 1997; EPPO 2017). A single 434bp fragment was obtained from Hezhang and Weining populations. A host test for the Hezhang and Weining populations were performed by inoculating 1,000 eggs per plant of varieties Qingshu 9, Huize 2 and Hezuo 88 grown in the pots containing 800 cm3 of sterilized soil (soil: sand ratio was 3:1), and four replications were tested in greenhouse under 16 h light, 22°C in the day and 8 h dark in the night. At 90 days post inoculation, 32.6 ± 7, 31.2 ± 8, and 29.5 ± 8 females and cysts were extracted from the infected roots and soils of the varieties Qingshu 9, Huize 2 and Hezuo 88, respectively. No females and cysts were observed on the control plants. The trial indicated that potato cultivars Qingshu 9, Huize 2 and Hezuo 88 are hosts for the Hezhang and Weining populations of Globodera rostochiensis. To the best of our knowledge, this is the first detection of potato cyst nematode Globodera rostochiensis in Guizhou Province, China.

The potato cyst nematode, Globodera rostochiensis, is a quarantine pest in Australia affecting a relatively small number of properties in three different production areas in the State of Victoria. The effects of susceptible (Trent, Sebago and Coliban) and H1‐resistant potato cultivars (Atlantic, Crop 13 and Nicola) on nematode populations were compared in two trials in a naturally infested field in the 2008/09 season (Trial 1) and in the 2009/10 season (Trial 2). The latter included a bare fallow treatment. The reproduction factor Pf/Pi (final compared with initial nematode population) was used to determine treatment effects. Initial population density was very high, averaging 111 and 119 eggs g−1 soil in Trial 1 and 2, respectively. The Pf/Pi of population density (eggs g−1 soil) was greater after growing susceptible cultivars (average 1.74 and 2.92 in Trials 1 and 2, respectively) than after growing resistant cultivars (average 0.72 and 0.61 in Trials 1 and 2, respectively), or after a bare fallow (1.09) in Trial 2. This correlated with higher Pf/Pi values of cysts 500 g−1 soil and eggs cyst−1 for susceptible cultivars than for resistant cultivars. Average Pf/Pi values greater than one in both trials are consistent with more cysts and an increased population density after growing susceptible cultivars. There was a trend of population decline, that is, Pf/Pi &lt; 1, after growing resistant cultivars (average Pf/Pi values of eggs cyst−1 of 0.77 and 0.35 and of eggs g−1 soil of 0.72 and 0.61, for Trials 1 and 2, respectively). However, Trial 2 showed that these Pf/Pi values were not significantly less than those for the bare fallow (0.68 eggs cyst−1 and 1.09 eggs g−1 soil). The susceptible cultivars Trent and Sebago produced lower yields than the resistant cultivars in both trials. In contrast, the susceptible Coliban yielded as well as the resistant cultivars, suggesting a high level of tolerance of this cultivar to infestation by the nematode. The resistant cultivar Crop 13 produced 34% and 36% greater total yields in Trials 1 and 2, respectively, than the once popular, but susceptible cultivar Sebago. This is the first report of the effects of potato cultivars on a population of the potato cyst nematode in an Australian field. The use of H1‐resistant potato cultivars can feasibly reduce populations of G. rostochiensis Ro1 on infested land and reduce PCN risk on land regulated as “linked” with infested land.

The potato cyst nematodes (PCN) Globodera pallida (Stone, 1973) and G. rostochiensis are key pests of potato, subject to strict quarantine regulations worldwide (EPPO 2013a). Indigenous to South America, they have spread to numerous potato-growing regions around the world. G. rostochiensis was reported from Kenya in 2015 (Mwangi et al. 2015). During a nationwide survey conducted in 2016, G. pallida was detected in Kenya at an altitude of 2,349 m above sea level in Nyandarua County (0.3150195° N, 36.48328° E). Cysts were extracted from a 200 cm³ soil sample following EPPO diagnostic protocol (EPPO 2013a), and then handpicked under a stereo microscope. The PCNs recovered showed morphometric characteristics of G. pallida and are reported here. For further studies, the Nyandarua field was resampled in February 2017 to collect additional soil samples and confirm the occurrence of G. pallida. From the collected cysts, 10 cysts were inoculated on potato (Solanum tuberosum L.) ʻShangiʼ in five pots with sterile soil and sand (1:1) and grown in a screenhouse for 3 months from May to July 2017; the multiplication rate at harvest was X― = 3.6 and PCNs were recovered from potato roots and soil. Morphometric characters showed: Granek’s ratio (n = 33) ranged from 1.53 to 4.52 µm (X― = 2.78 ± 0.78 µm), and the distance from anus to vulval basin was 34.03 to 91.45 µm (X― = 52.75 ± 13.73 µm). The stylet length of the second-stage juveniles (J2s) (n = 97) ranged from 15.87 to 25.18 µm (X― = 21.87 ± 1.43), stylet knobs displayed robust tulip/anchored shape. The lengths of the hyaline tail (HT) and the true tail (TT) ranged from 15.54 to 50.44 µm (X― = 23.94 ± 4.23) and 31.02 to 79.59 µm (X― = 50.64 ± 5.71 µm), respectively. Body length (n = 40) fluctuated from 338.41 to 468.34 µm (X― = 432.23 ± 24.95). DNA amplification was performed from 14 cysts and 25 J2s using the multiplex-PCR method adapted from Bulman and Marshall (1997) and the ITS1-5.8S-ITS2 regions (Tirchi et al. 2016). PCR cycling parameters were adjusted to a 5-min initial denaturation phase and 37 PCR cycles for multiplex-PCR (EPPO 2013b). The species-specific primers ITS5/PITSp4 for G. pallida (265 bp) and AB28/TW81 primers (1,188 bp) were used to amplify the small subunit of the 18s ribosomal RNA and the ITS region, respectively; PCR-amplicons were purified using the QIAquick PCR Purification Kit (Qiagen, U.S.A.) and the DNA sequences were manually edited using BioEdit Sequence Alignment Editor; in silico analyses were conducted with the NCBI-BLAST tool. The Kenyan ITS5/PITSp4 sequences (NCBI accession no. MG309873) presented 100% similarity to the G. pallida isolates KJ409623.1 and AF016869 (score = 481; E value = 5.02e⁻¹³²), while the Kenyan AB28/TW81 sequence (NCBI accession no. MG309920) showed 95 and 94% similarity to the G. pallida isolates HF583248.1 and HQ670272.1 (score = 1,218 and 1,221; E value = 0), respectively. This first report of G. pallida in sub-Saharan Africa has paramount phytosanitary and regulatory implications for potato growers and traders, national extension services, and policy makers in Kenya and the surrounding region.

Genetic variation in potato cyst nematodes in Northern Ireland

SUMMARYThe effect of a pre‐planting application of oxamyl on the yields of six potato cultivars was studied in co‐operative field trials in 1981. Two sites were ‘uninfested’ with potato cyst nematodes (PCN), two were lightly infested (&lt;25 eggs/g soil) and six were moderately to heavily infested (three with Globodera rostochiensis and three with G. pallida). At the uninfested and lightly infested sites oxamyl had little effect on mid‐season haulm weights or on final tuber yields. At sites moderately to heavily infested with G. rostochiensis the haulm growth of all cultivars tended to be increased by a similar amount on plots treated with oxamyl, Pentland Dell being least responsive. Yield was increased by different amounts, the increases being least for cvs Cara and Maris Piper and most for Corsair and Pentland Dell. At the sites moderately to heavily infested with G. pallida Cara was again tolerant, its yield being increased little by oxamyl compared with the other cultivars. Maris Piper gave the largest yield increase.Final populations of PCN on non‐resistant cultivars were reduced by oxamyl at some sites but not at others. Resistant cultivars also decreased the final numbers of PCN at most sites. Two cultivars derived from Solanum vernei with different degrees of resistance, appeared to be almost equally effective in controlling G. rostochiensis and G. pallida.