A new method of quantitative detection of Chalara elegans and C. thielavioides in soils using carrot discs
Werner E Heller
Research Station Agroscope Changins-Wädenswil ACW, CH-8820 Wädenswil, Switzerland werner.heller@acw.admin.ch
Received 25 June 2012, accepted 25 September 2012
Abstract
A new technique for the quantitative detection of Chalara elegans and C. thielavioides in soil based on the combination of a soil dilution technique with carrot disc baits was devel- oped. The quantitative Chalara test was validated in repli- cated experiments using mineral and organic soils in our laboratory. The test allowed detection of a population den- sity of 1 cfu 0.1 g–1 fresh soil. Efficiency, repeatability and dependability of the test were regarded as acceptable and this was confirmed by an international ring test with 7 par- ticipating laboratories. With the quantitative Chalara test, the influence of crop cultures or green manures on the population densities of C. elegans and C. thielavioides in the soil can be assessed.
Key words: Thielaviopsis basicola, population density
Introduction
Chalara elegans (synanamorph: Thielaviopsis basicola) is a ubiquitous soil-borne, plant pathogenic fungus found in agricultural and non-agricultural soils (Yarwood 1974).
Chalara thielavioides (syn. Chalaropsis thielavioides) is an- other related fungal species. Both fungi are pathogens of carrots causing in the last few years severe quality prob- lems of carrots at the points of sale in Switzerland (Heller 2000) and in other European countries (Weber & Tribe 2004).
In 2007, a survey of 650 stored, unwashed carrot samples provided by 6 processing facilities in northern Switzerland showed that 350 of them were infected by Chalara species.
C. thielavioides was detected on 70% of the infected carrot samples, C. elegans on 24% and both pathogens together on 6% (Kägi et al. 2007). In addition to crops of tobacco, beans, cotton and peanuts where C. elegans causes a serious black root rot, the fungus was found associated with a range of more than 130 cultivated and wild plant genera (Yarwood 1981). The significant impact of C. elegans on crop produc- tion has led to an extensive study of this organism. Since Yarwood (1946) described the isolation of C. elegans from soil or diseased roots using carrot discs, several methods for qualitative or quantitative detection of this pathogen in soils have been described (Punja & Chittaranjan 1994).
As C. elegans is not a competitive saprophyte (Hood &
Shew 1997), the soil dilution techniques to detect its pres-
ence have to resort to elaborate selective media composed of nutrients, fungistatic and bacteriostatic substances to suppress the development of the more competitive sapro- phytes. These selective media can be as sensitive as carrot discs but such a test is complicated, time consuming and costly (Specht & Griffin 1985). The use of carrot discs to isolate C. elegans from infected tissue has a long tradition with plant pathologists and the astonishing specificity of the carrot bait for the pathogen has been emphasised (Tabachnik et al. 1979). The population densities of C. elegans men- tioned in earlier investigations using streptomycin sulphate amended soil suspensions and a carrot disc sandwich tech- nique or artificial media are considerably higher than one colony forming unit (cfu) cm–3 soil, or 1cfu g–1 soil, indicat- ing a limit of detection of this order (Rittenhouse & Griffin 1985, Specht & Griffin 1985).
The objective of the present paper is to describe a new quantitative method to detect Chalara elegans and C. thiela- vioides in soils using carrot discs as a selective medium.
Materials and methods Production of carrots and discs
To avoid contamination with soil-borne C. elegans, carrots (Daucus carota, cv. Bolero) were produced from seeds dis- infected with aerated steam (at 65°C for 90 sec.) in a pas- teurised mixture of 70% peat and 30% perlite in raised beds (5 × 1 m). Carrots were harvested, washed with tap water, dried on blotting paper on a sterile bench and cut into 4-5 mm thick discs under hygienic conditions. For each soil sample or dilution step 50 carrot discs were placed in a new or disinfected plastic box (15 cm × 20.5 cm × 5 cm) to be used later during the incubation.
Soil sampling procedures and preparation of soil suspensions
At least 30 cores of 20 cm deep topsoil per plot were taken randomly with a 25 mm diam. Edelman auger and collected in a fresh plastic bag. In our experimental farm with its limited plots, a history of 40 years of vegetable production with a rotation of many Chalara-susceptible crops, strict weed control and frequent and intense tillage, an even dis- tribution of the pathogen in the topsoil layer can be assumed and so the soil could be sampled randomly.
Soil samples were dried in the opened bags at room temperature until they could be worked on, then passed through a sieve (4 mm mesh size, autoclavable, stainless steel) to mix the soil and to remove gravel and stones and then stored for at last 3 weeks in closed plastic bags at 3°C to reduce biological activity in the soil before starting the test.
All the soils tested were slightly alkaline (pH 7-8). The first two soils (Cherry tree and Terra Alta) were sandy loams but the last four soils were rich in organic substance or organic soils.
To prepare a quantitative Chalara test, 10 g of a sieved soil sample were mixed with 90 ml water agar (0.15%, Difco), and the resulting 10–1 diluted suspension stirred at room temperature for 60 min.
Inoculation of the carrot discs and incubation
A sterile 1 ml syringe (Becton Dickinson Plastipak) was filled with the 10–1 soil/water agar suspension whilst stirring.
Diluted water agar was used to prepare soil suspensions in order to prevent sedimentation of soil particles and fungal inoculum in the syringe during the inoculation procedure.
Using the repetitive pipette (Tridak stepper™), each car- rot disc was inoculated with one 20 μl droplet of the soil/
agar suspension. Thus, a total of 0.1 g of fresh soil was distributed amongst the 50 carrot discs placed in suitable small plastic boxes. After inoculation they were laid out in larger plastic boxes containing a wet paper towel to act as damp chambers during incubation for 10 days at 20°C in the dark.
Assessment and determination of population density of C. elegans or C. thielavioides
After incubation, the points of inoculation on the carrot discs were inspected under a dissecting microscope equipped with a 40 × magnification for mycelial growth and chlamydospores of both C. elegans and C. thiela- vioides. Only inoculation points with distinctly visible chlamydospores were assessed as positively infected by a Chalara spp. Since the chlamydospores of each species are quite distinct, it would be easy to assess their popula- tions individually. However, they both cause the same symptoms on carrots and to simplify the international ring test, the species were not separated in these experi- ments.
When more than 20 of the 50 inoculation points were found to be positive for Chalara spp., the test was repeated with a fresh soil suspension in a 10–2 dilution with water agar (0.15%).
The population density (P) of Chalara spp., expressed as the number of cfu 0.1 g–1 fresh soil (FS) can be counted directly as the number of droplets positively infected by Chalara spp., in the 50 droplets per soil sample. To express P as cfu kg–1 fresh soil, multiply the figure by 104.
International ring test
Six foreign laboratories, most of them delegating members to the European Mycological Network (EMN), agreed to take part in the ring test. The Belgian laboratory did two tests: one with carrots free of Chalara spp. delivered by ACW (Agroscope Changins-Wädenswil) and one with fresh local market carrots. Other laboratories used only the Chalara- free carrots supplied by ACW and were situated in Lithuania, Spain, Slovenia, and two in Germany. The laboratories were sent a detailed description of the quantitative Chalara test (QTC)-manipulations before agreeing to take part in the ring test.
Five soil samples were taken randomly from different plots of the ACW experimental farm “Sandhof”, prepared as described and sent to each laboratory in batches of 100 g together with a sufficient amount of test carrots free of Chalara spp.
Duplicate soil samples used in the ring test were tested in the ACW laboratory with 4 fold replication and the results compared with those from the participating laboratories.
A statistical analysis (Duncan test) performed with the WIDAS software of the data represented in Table 1 revealed no significant differences (p < 1%) amongst the individual laboratories indicating that they all produced comparable results.
Results
Intra laboratory replications of the test at ACW
As a first stage the repeatability of the QCT was examined over a time period of several months. At least three different persons carried out replications of the test in our laboratory.
The range of soils tested here included slightly alkaline, sandy loams and organic soils from different locations of the Swiss plateau. The Results are presented in Fig. 1.
The investigations repeated by different persons showed that the repeatability of the test is satisfactory since the determined population densities varied within acceptable limits. There was no obvious effect of soil type on the sensi- tivity of the test.
Efficiency of QCT
The efficiency of the quantitative Chalara test was assessed by dilution of the vital inoculum in a naturally infested soil with an autoclaved subsample of the same soil. With this technique the density of the vital inoculum in soil samples could be reduced without significant modification of the composition of the soil suspension. Samples containing mix- tures of 0, 25, 50, 75 and 100% untreated soil with auto- claved soil were tested with QCT. The experiment was carried out in 4 replications at intervals of 3 to 4 weeks. For each replication the correlation of inoculum concentration in the soil mixture with the Chalara population density detected by QCT was calculated by Excel®: r = 0.9898, 0.9345,
0.9931 and 0.9490. In Fig. 2 the detected population den- sities of an exemplary replicate is shown.
International Ring Test
In this test all participants detected comparable Chalara population densities in the individual sandy loam soil sam- ples collected in ACW’s experimental farm “Sandhof”. The plots tested were integrated in different rotations and were therefore suspected of harbouring different population den- sities of Chalara spp. as is indeed shown by these results.
All the values per tested plot were in the same order of magnitude. None of the reported results had to be discarded as an obvious outlier. The results of this test are given in Table 1.
Discussion
The data presented here show that under practical field conditions the new quantitative Chalara test detects with high reliability Chalara populations in soil samples from vegetable production areas. The high correlations demon- strated in the efficiency evaluation between the inoculum densities detected by QCT and the varying concentrations of vital inoculum in soil samples confirm that the inoculum is detected quantitatively.
In some situations, as outlined by Rittenhouse & Griffin (1985), soil sampling procedures should be adapted to the prevailing cultural, topographic and edaphic conditions, because the distribution of the fungi in the soil can be aggre- gated and more elaborate sampling schemes should be observed.
Table1:Data, expressed as cfu 0.1 g–1 fresh soil sample, generated in the international ring test as reported by the partici- pants. Means, variances and standard deviations have been calculated by ACW.
Sample no. 1 2 3 4 5
Weight of fresh soil tested 0.1 g 0.1 g 0.1 g 0.1 g 0.1 g
Participant
ACW (mean of 4 replicates) 14.3 5.5 28.8 11.3 6.5
Monika Heupel (Germany) 17.0 8.0 50.0 20.0 13.0
Hermann-Josef Krauthausen (Germany) 8.0 3.0 19.0 15.0 9.0
Alenka Munda (Slovenia) 12.0 7.0 16.0 10.0 6.0
Ana Maria Perez Sierra (Spain) 9.0 7.0 11.0 7.0 5.0
Zita Jovaisiene (Lithuania) 11.0 7.0 26.0 1.0 11.0
S. Inghelbrecht (Belgium, mean of 2 rep.) 9.0 5.5 19.5 16.0 17.0
Mean 11.46 6.14 24.32 11.46 9.64
Standard deviation 3.10 1.45 9.30 5.51 4.87
Variance 3.24 1.65 12.78 6.29 4.33
Fig. 1: Mean values of Cha- lara species (C. elegans and/
or C. thielavioides) population densities (cfu 0.1 g–1 fresh soil) in soil samples of several prov- enances in quantitative Cha- lara tests repeated over time.
1, Cherry tree, sandy loam;
2, Terra alta, sandy loam; 3, Haslen 5, sandy loam; 4, TAW 17, organic soil; 5, TAW 9Z, organic soil; 6, TAW 9P, organic soil. The number of replica- tions of the test is indicated in brackets. The error bars in- dicate standard deviations of the values.
Under favourable conditions infection of carrots by Cha- lara spp. can occur at any time, during growth in the field, harvest, storage, washing or packaging processes. Since these conditions are intrinsically very variable, in practice there can unfortunately be no reliable correlation between the Chalara population density in the soil and the potential occurrence of carrot black root rot as a quality problem at the point of sale.
It is important to note that in recent years carrots were produced on most of the plots tested in the international ring test, but carrot black rot caused by Chalara spp. was never observed. It can therefore be assumed that Chalara population densities of less than 25 cfu 0.1 g–1 can allow carrot production to be free of black root rot at the point of sale, provided that carrots are cooled rapidly to 1°C, then kept under constant storage conditions and finally washed and intensely rinsed exclusively with fresh water.
If carrot storage and processing conditions are not opti- mised, especially if the product comes into contact with processing water contaminated by Chalara spp., the value of a low threshold of soil inoculum density is greatly re- duced because post harvest infection has a much greater impact on the incidence of Chalara black root rot at the point of sale.
With the proposed dilution of 10 g fresh soil in 90 ml water agar, the detection limit of the described QCT is in theory 1 cfu 0.1 g–1 of fresh soil, which is less sensitive than the detection limits mentioned by earlier authors (Tabachnik et al. 1979) but by changing the dilution ratio and by distributing more or larger inoculum droplets on a greater number of carrot discs the detection level of the test can be easily increased. The QCT does not require any special preparation procedures of the test soils or the use of antibiotics. In general carrots used for the test should be produced under Chalara free conditions. But the QCT can even be performed using market carrots washed thoroughly with fresh tap water before use if non-inoculated control carrot discs are included in the test series.
The deviations of the reported results in the international ring test were regarded as acceptable as this was the first time that most of the participants had done the test.
In terms of a soil biology experiment the quantitative Chalara test (QCT) can be regarded as quite robust in view of the relatively low intra- and inter-laboratory deviations.
With the quantitative Chalara test, the influence of crops or green manures on the population densities of Chalara spp. in soils can be investigated and also soils can be tested before Chalara susceptible perennial crops like cherries or other stone fruit trees are planted. Thus heavily contami- nated soils can be avoided and the risk of black root rot in crops reduced.
The QCT can be adapted to monitor Chalara contamina- tions along the food processing chain where this seems appropriate. The author is aware that, to reach a detailed interpretation of the significance of Chalara inoculum levels in soils, a wider study of Chalara population densities in naturally contaminated sites for comparison with disease severity of host plants remains to be undertaken.
Acknowledgements
The participation of the following European Mycological Network members in the QCT-ring test is gratefully acknowl- edged: Zita Jovaisiene (State Plant Protection Service, Phytosanitary Research Laboratory, Pelesos str. 85, 2014 Vilnius, Lithuania. (EMN); Ana Maria Perez Sierra (Insti- tuto Agroforestal Mediterraneo, Universidad Politecnica de Valencia, C/Camino de Vera s/n, 46022 Valencia, Spain.
(EMN); Alenka Munda (Agricultural Institute of Slovenia, Hacquetova 17, 1001 Ljubljana, Slovenia. (EMN); Monika Heupel (Landwirtschaftskammer Nordrhein Westfalen, Pflan- zenschutzdienst, Siebengebirgsstrasse 200, 53229 Bonn, Germany. (EMN); Sven Inghelbrecht, Kurt Heungens (Insti- tuut Voor Landbouw- en Visserijonderzoek (ILVO), Eenheid Plant – Gewasbescherming, Burg. Van Gansberghelaan 96 bus 2, 9820 Merelbeke, Belgium. (EMN); Hermann-Josef Krauthausen (Gruppenleiter Phytomedizin-Gartenbau, DLR Rheinpfalz, Breitenweg 71, 67435 Neustadt/Weinstrasse, Germany).
The author thanks Roger T A Cook, Consultant Plant Pathologist and Secretary of the European Mycological Network, 30 Galtres Avenue, YORK. UK, for the revision of the manuscript.
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