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Development of a Nordic system for measuring the inactivation of pathogens during composting

3. Indicator organisms for plant pathogens

3.1 Purpose

The purpose of this part of the investigation was to:

1. Collect or produce material infected with the pathogens Plasmodiophora brassicae, Fusarium oxysporum, Rhizoctonia solani, and tobacco mosaic virus for the direct process evaluation.
2. Survey the possibility for complementary microbiological techniques (ELISA) in the evaluation of biotests
3. Perform biotests with the selected pathogens and compared results obtained visually and with ELISA techniques.
4. Investigate the resistance of the organisms under standard conditions.

3.2 Materials and Methods

3.2.1 Plasmodiophora brassicae

Inoculum were collected in a red cabbage field in December 1999 at a farm in the vicinity of Skælskør. Infested soil and gall material was air-dried for about 10 days, soil and gall material was ground with mortar and pestle and passed through a 10 x 10 mm mesh.

The inoculum (30 g gall material and 430 g infested soil) was mixed with a sand-clay-peat mixture, using 30% sand (volume) and 70% Weibull enhetsjord (K-jord) clay-peat (20%:80% by volume) mixture, amended with N, P, K and trace elements by the manufacturer (Weibull Trädgård AB, Box 520, 261 24 Landskrona, Sweden). A sufficiently low pH (£ 6.0) was obtained by mixing the inoculum with approximately 550-mL of sand-clay-peat mixture in the specified ratio.

In all biotests for P. brassicae, incubation of treated plants took place in the greenhouse with 16 h light at 24° C alternating with 8 h darkness at 17?C. A liquid fertiliser (Hornum Næring, P. Brøste A/S, Lyngby, Denmark) was used for maintenance of plants at a rate of 20 mL fertiliser in 4 L water.

Based on the guidelines formulated in Kehres & Pohle (1998) the mustard cultivar ‘Vittasso’ of Brassica juncea (L.) Czernj. et Cosson was chosen for the biotests.

A five step soil dilution series (dilution factor 2) was prepared in three replications, using an inoculum level of 30 g club-root material and 430 g infested air-dried soil at the first dilution step in a mixture with 200 g sterile sand and 550 mL sand-clay-peat mixture. This composition yielded a mixture having a satisfactory low pH (< 6). In subsequent dilution’s, inoculum volumes were replaced with equal volumes of sand-clay-peat mixture. Hence, the dilution steps contained 1, 0.5, 0.25, 0.125 and 0.0625 times of the basic inoculum mixture (30 g club-root material and 430 g infested soil). The samples were placed in 2-L pots (13-cm diameter). 16 seeds of B. juncea, cultivar Vitasso, were sown in each pot, water was supplied (300-500 mL per pot) and pots were packed in polyethylene bags, in order to avoid cross-infections between pots. Extra seeds of B. juncea were germinated on moist blotter, and after one week, seedlings were transplanted to pots in which seeds had not germinated in order to assure a total of 16 plants per pot. A pathogen free control pot in which the inoculum volume was replaced by sand-clay-peat mixture was sown, and all pots were then incubated in the greenhouse.

The B. juncea seedlings were observed frequently, and since pronounced club-root formation was seen after 14 days of incubation results were scored at this time, recording plants as either club-rooted or not. Root samples without evident symptoms were selected at random, prepared as whole mounts in water and examined at magnifications of 10 x 10 - 40 in light microscopy.

Club-root development over time. Twenty-two samples were prepared (30 g club-root material, 430 g infested soil, 200 g sterile sand (autoclaved twice at 121?C, 1.5 atm. for 1 h at a 24 h interval), wrapped in polypropylene fibre tissue (Windhager ‘Garten-Vlies’) and buried in moist, sterile sand (moisture content 20-25%) in a polyethylene sealed tray. The samples were incubated for 28 days at room temperature and then 18 samples were transferred to 2-L 13-cm diameter pots after mixing each sample with 550-mL sand-clay-peat mixture. 16 pre-germinated seedlings (7 days old) were transplanted to each pot. Three replicate pathogen free pots (16 plants per pot) were produced as described above for control. Disease was scored in sets of three replicate (treated) pots after 18, 28, 35 and 42 days of incubation. Roots of symptomless plants were subjected to microscopy as described above.

Soil dilution series. Combining two samples, the remaining four samples were used to make two replicate soil dilution series, the initial dilution step representing one sample, and the following, as above 0.5, 0.25, 0.125 and 0.0625 times the full sample dosis. Sixteen mustard seedlings (one week old) were transplanted to each pot. Three inoculum free pots were planted with 16 plants in each to serve as reference. Disease scoring took place after a full 6-week incubation period in the greenhouse.

Disease scoring for the two above assays was made using a four step disease index, modified after Buczacki et al. (1975), in which 0 = no visible root swelling, 1 = symptomless roots but plasmodia present / slight root swellings, 2 = moderate and 3 = large root swellings. Unlike the index suggested by Buczacki et al. (1975), the applied index did not consider whether lateral and / or taproots were infected, since swellings were consistently associated with the taproot. Further, the results of the study of club-root development over time were analysed as discrete data in ordered response categories by logistic regression in SAS System 6.12.

3.2.2 Rhizoctonia solani

A Danish isolate, accession number 2041, of Rhizoctonia solani, AG1, which has proven pathogenic to bean (Phaseolus vulgaris) was propagated on wheat kernels to form the amount of inoculum (10 kg) required for the pilot and full-scale investigations. Wheat seed in 2-L Erlenmeyr flasks, 750 g in each, was mixed with 625-mL water per flask and twice autoclaved for 60 minutes at a 24 h interval. The content of each flask was inoculated with 50 mL of a homogenate of 700 mL water and 10 5-day-old colonies of R. solani on potato dextrose agar (1.5% PDA, Difco in 90 mm Petri dishes). A 14 days incubation period at 20?C followed, after which 25 samples, each equivalent to 30 g dry seed, were wrapped in polypropylene fibre tissue (Windhager ‘Garten-Vlies’) and incubated for 28 days at room temperature in moist sterile sand (20-25% moisture by weight). The surplus inoculum was air dried in open trays in a laminar flow bench at 20?C, until a weight of less than 2 kg per bag had been reached - about 5 days were required. The inoculum thus produced was again transferred to sterile autoclave bags, which were heat sealed and stored at 4?C.

Survival of the pathogen was tested by plating a few wheat seeds from each of the incubated samples on 1.5% water agar and incubating for 2-3 days, after which observations were made in low-power light microscopy for the characteristic mycelium and anastomoses of R. solani.

A preliminary soil dilution test (three replications) was carried out under the same temperature and light conditions as applied for the P. brassicae tests (dilution factor 2, 5 steps, initial dosis 30 g) in order to check the potential of the produced inoculum. A single, inoculum free pot was sown for reference.

Further, in the same design, triplicate soil dilution series (initial dose 3 g per pot, dilutions 1, 0.2 and 0.1) were incubated in the light / darkness conditions described in 3.2.1.3, in low, medium and high temperature regimes (day / night: 15 / 17; 17 / 24 and 25 / 28?C) to test in which regime the biotest would perform the best. Triplicate inoculum free pots were sown for reference in all temperature regimes.

The dwarf bean (Phaseolus vulgaris) variety ‘BonBon’ was used as bait plant. In the preliminary test the sowing rate was 16 seeds, and in the soil dilution series test 20 seeds, per pot (1-L, 13 x 11.5 cm rectangular pots). The growth medium was a 30% sand : 70% clay-peat mixture, and the sown seed was covered with a mixture of the basic sand-clay-peat medium and the amount of inoculum required for the specific test performed.

The incubation period was three weeks in all tests. Plants were maintained using Hornum fertilizer. At the end of the three-week incubation period disease incidence was recorded. In the preliminary test a simple index was used, in which 0 = no symptoms, 1 = canker lesions present and 2 = seed rot, pre- and post-emergence damping-off. The soil dilution test involved an index in which 0 = no symptoms, 1 = small to medium size canker lesions on either hypocotyl or on root 2 = large size canker lesions on either hypocotyl or on root 3 = canker lesions on both hypocotyl and on root and 4 = seed rot, pre- and post-emergence damping-off. The data were analyzed by logistic regression in SAS System 6.12 (Proc Probit). The latter index was modified by pooling of neighbour categories as appropriate (See results).

A Rhizoctonia spp. specific ELISA kit, Agri-Screen, was purchased from Neogen Corporation, Lansing, UK. Segments of 2-cm length were cut from the root-hypocotyle transition of all plants in the soil dilution series incubated in the three temperature regimes.

The segments were bulked, weighed, and homogenised in test tubes with the extraction buffer (4 ml) provided in the kit, using an Ultra Thurax blender. Duplicate wells were filled with sample extract from each of the 35 pots from which plants could be harvested, out of a total of 36 pots involved in the experiment (no plants emerged in pot no. 36). A positive (pure, sterile R. solani extract) and a negative standard reference in single wells were included in the test. After this step, the procedure specified by Neogen was followed.

3.2.3 Fusarium oxysporum

A Danish isolate, accession number 2065, of Fusarium oxysporum f.sp. lycopersici, race 0, which has proven pathogenic to tomato (Lycopersicon esculentum) was propagated in 6 kg wheat seed, according to the procedure as described for R. solani (inoculum per flask: 50 mL of a homogenate of 400 mL sterile water and 6 5-day-old F. oxysporum on PDA). At the end of the incubation 22 samples equivalent to 30 g dry seed each were wrapped in polypropylene fibre tissue and buried in moist sand (20-25% humidity by weight) for the four week incubation. The remainder inoculum was air dried and stored at 4?C for the full-scale investigation, as was the R. solani inoculum.

A few seeds from each incubated sample were plated on plain 1.5% water agar and incubated at 20?C for 2-3 days, followed by observations for Fusarium growth after 3-4 days.

A sand(3):fine(1):medium(1) vermiculite mixture was used as growth medium. The susceptible tomato (Lycopersicon esculentum) variety ‘Harzfeuer’ was used as bait plant in the biotests. Dilution series of steps 1 (initial step 30 g inoculum), 0.2, 0.1, 0.025 and 0.01 were made in triplicate in three glasshouse temperature regimes, as applied for R. solani tests. Inoculum free pots were sown in triplicate for each temperature regime for reference.

Pots were inoculated by placing a 150 mL mixture of sand:vermiculite and inoculum as required on 500 mL of the basic sand:vermiculite mixture, and the inoculum layer was then covered by a 100 mL sand:vermiculite layer, in which 20 seeds were sown. Plants were maintained using Hornum fertilizer. Plants were incubated for 10 weeks.

3.2.4 Tobacco mosaic virus

An isolate of tobacco mosaic virus was obtained from Department of Plant Protection, Danish Institute of Agricultural Sciences. This isolate was originally obtained from ATCC as isolate PV135. The virus was propagated in tobacco plants (Nicotiana tabacum var. Samsun or Wisconsin 38), where it spreads systemically. Plants at the four- to five-leaf stage were manually inoculated by applying sap from infected tobacco plants, diluted approx. 1:10 in inoculation buffer (4% polyethylene glycol 6000, 30 mM sodium phosphate pH 7.7), onto leaves previously dusted with carborundum as abrasive. Plants were grown under normal greenhouse conditions. Two to four weeks after inoculation, leaves showing mosaic-type symptoms were harvested and packaged in gauze.

Three samples each containing 10 g of infected tobacco leaves were placed in approx. 1 kg of wet sand each in plastic bags and incubated for 31 days at room temperature.

After incubation, each sample (consisting of the leaf material and part of the gauze packaging to which it stuck) was placed in a mortar together with approx. 10-mL inoculation buffer and pounded with a pistle. The resulting slurry was inoculated to tobacco plants (Nicotiana tabacum var. Samsun or Xanthi-nc) as described above. Three Samsun plants and 1-5 Xanthi-nc plants at the three- to four-leaf stage were inoculated for each sample. Two leaves were inoculated on each plant. 12 days later the plants were visually inspected, and samples of inoculated (Xanthi-nc) or systemically infected (Samsun) leaves were analysed by ELISA

0.25 g leaf material was homogenised in 2.5 mL 50 mM phosphate buffer pH 7.4 containing 140 mM NaCl and 0.1% Tween-20. The samples were analysed by the standard double-antibody sand with ELISA technique (Clark and Adams, 1977) using anti-tobacco mosaic virus reagents from BIOTEBA AG (Schweiz; Art. No. 190415 and 190425).

3.3 Results

3.3.1 Plasmodiophora brassicae

Club-root symptoms developed within the 14 days of incubation in 167 of a total of 197 plants. Microscopy of roots randomly sampled from the 30 symptom free seedlings showed the presence of P. brassicae plasmodia in the taproot. A random variation of disease levels rather than a systematic relationship between these levels and the applied dilution steps was obvious in the three series (Table 1). There were no club-root symptoms in the control plants. Despite transplanting, the number of seedlings surviving at the end of the incubation period was less than 16 in many pots, due to poor plant establishment.

Club-root development over time. Club-root symptoms were evident in the majority of mustard seedlings already after 14 days of incubation, and pathogen survival was verified for all twenty-two incubated test samples, since symptomatic plants were found in all pots, except for the control pots. While tap root swelling was consistently seen in all symptomatic plants, swelling of lateral roots was only found in some plants after 34 and 41 days of incubation.

Disease scoring was made in sets of three replicate pots on days 18, 27, 34 and 41 after transplantation, and showed a gradual increase in severity over time (from 2.04 to 2.88) as may be seen in Table 2. Odds-ratio analysis of the data revealed that only the disease level observed on the earliest date was significantly lower (P = 0.0001) than the disease level observed after 6 weeks of incubation, indicating that - at least at the very high inoculum level applied here - the biotest could be terminated earlier than the 6 weeks prescribed for the test.

All asymptomatic plants were subjected to histological examination. In 18 and 27 days old plants microscopy of tap roots as whole mounts was unproblematic, since the tap roots were almost fully transparent, while observations in older plant roots were less readily obtained, due to decreased transparency. For a number of plants scored to index level 1 infection was

Table 1.
Number of mustard plants (Brassica juncea, variety ‘Vitasso’) with club-root symptoms (Plasmodiophora brassicae) relative to total plant number in three replicate soil dilution biotests, following a 14 days incubation period in greenhouse.

Table 2.
Development of club-root disease (Plasmodiophora brassicae) severity in mustard seedlings (Brassica juncea) over time.

^**Disease index = ?(n_i x i)/N, where i = numerical value of index group designation, n_i = number of plants in corresponding index group i and N = total number of plants in pot.

only visible in microscopy. Histological examination revealed infections in all but a single of the asymptomatic seedlings (Table 2).

Soil dilution series. As in the preliminary tests the disease score results of the duplicate soil dilution series made from incubated samples revealed no difference in effect of different inoculum levels on the disease levels (Table 3).

Table 3. Development of club-root disease (Plasmodiophora brassicae) in mustard seedlings (Brassica juncea) inoculated at five inoculum levels (soil dilution factor 2) after 6 weeks of incubation. First dilution step = 30 g club-root material and 430 g infested soil.

^**Disease index = ?(n_i x i)/N, where i = numerical value of index group designation, n_i = number of plants in corresponding index group i and N = total number of plants in pot.

3.3.2 Rhizoctonia solani

After 2-3 days of incubation, characteristic growth of R. solani was observed in water agar platings of material from all incubated samples, and no contaminating organisms were observed in the plated material. Hence, the pathogen survived the incubation process without problems.

Typical symptoms were ellipsoidal, sunken, red-brown to very dark cankers on root and hypocotyl. Mycelium was often found to proliferate over the hypocotyl. Epidermal reddish-brown fissures were extremely frequent in both treated and in reference pots, and it was found that the discolouration was due to necrotic epidermal tissue remnants suspended in the fissures. While R. solani often colonised such fissures, it was obviously not the cause of their formation. In any case of doubt, it was verified by stereomicroscopy that R. solani mycelium was present in lesions, before scoring a plant as infected.

In the preliminary test seed rot and pre- and post-emergence damping-off were pronounced at high inoculum levels. Since an extremely high incidence of seed rot and pre- and post-emergence damping-off was observed at the highest inoculum level in the preliminary test it was suspected that phytotoxic metabolites produced by the pathogen were responsible for the observed seed and seedling death. For that reason a special assay was designed to examine this, which confirmed the suspicion (data not presented). In consequence, the highest inoculum level in the soil dilution tests was reduced to 0.1 of that applied in the preliminary test, and the dilution scale widened considerably.

No disease was observed in the reference pot in the preliminary test, and as could be expected, in the statistical analysis all treatments were significantly different from the reference. When considering only comparisons between inoculum levels in treated pots in the statistical analysis, significant difference in disease incidence was only found for the comparison of the two extreme dilutions (P = 0.0014, level of significance 0.01).

In the soil dilution series tests, the index level 2 had a very low frequency, and it was most appropriate to pool this category with the higher level 3, obtaining thus a four-category index. Analysis showed that when corrections for overdispersal was made, there was no significant effect of replications or of temperature regimes, while all treatments significantly differed from the reference group. In this analysis there was no significant difference between treatments (inoculum levels).

Although no statistically significant effect of different temperature regimes was revealed, it was noted that plant emergence and disease development were better / more severe in the highest temperature regime, while disease frequency was slightly higher in the medium temperature regime than in other regimes.

Evaluation of the finished test by naked eye clearly showed that wells holding reference plant extract compared well to the negative standard (light pale green), while most wells holding extract from treated plants stained strongly green (some stronger than the positive control), which compared well to the positive standard. Wells representing treatments showed a wide colour tone variation, however, which obviously was partly due to the variable amount of homogenate used as basis for extraction, and which could be possibly related to the inoculum level background.

3.3.3 Fusarium oxysporum

Survival of F. oxysporum was confirmed in all incubated samples since the pathogen emerged from all material plated on water agar.

Specific chlorosis, epinasty and wilt symptoms started to appear after 10 weeks of incubation at all dilution steps, indicating a good sensitivity of the test, while reference plants appeared healthy. Reduction of plant emergence and height were obvious results of increasing inoculum levels. It was obvious that symptom development and disease severity increased with increased temperature. As for R. solani preliminary experiments were made, which showed that phytotoxic metabolites from the inoculum were capable of inhibiting plant emergence at the highest inoculum level (data not presented).

Due to the tardive development of symptoms, the results of the disease scoring cannot be presented in this report, since the scoring is presently being made.

3.3.4 Tobacco mosaic virus

The tobacco variety Xanthi-nc carries the N gene providing hypersensitive resistance against tobacco mosaic virus. All leaves inoculated with sample extracts showed distinct local lesions within a week. At the time of scoring (day 12 after inoculation), all inoculated leaves contained numerous lesions, > 100 on all leaves except three, which were seriously damaged by the inoculation. All Samsun plants showed mosaic symptoms on younger leaves.

All leaf samples were strongly positive in ELISA. At the time of reading (80 minutes after substrate addition), all sample wells showed the maximum value (absorbance at 405 nm) of approx. 4.0 as did the positive control sample (a tobacco mosaic virus infected leaf sample from the greenhouse), while the uninfected control sample showed a value of 0.403. A small Xanthi-nc leaf sample comprising only three local lesions also showed the maximal Abs(405) value at 80 minutes.

3.4 Discussion

3.4.1 Plasmodiophora brassicae

In the perspective of testing the capacity of sanitation plants to reduce or eradicate P. brassicae from pure compost both the applied inoculum concentration (as prescribed according to German guidelines (Kehres & Pohle,1998)) and the quality of the sampled inoculum were considered adequate in terms of posing a challenge to the various sanitation plants selected for the full-scale project. The preliminary soil dilution biotests confirmed that the inoculum vigour was high, the inoculum giving rise to visible symptoms in more than 80% of the plants after only 14 days of incubation, and in the test of incubated samples all plants were infected after 18 days of incubation.

Although the assay was made as a five step soil dilution series, the dilution factor never reduced the inoculum level below the level of detection, nor did the lower levels reveal any reduction of levels of attack as compared to the maximum inoculum concentration level. The four week moist sand incubation period had no detectable effect on the vigour of the inoculum.

P. brassicae could be detected prior to symptom development in roots of B. juncea (‘Vitasso’) plants after three weeks of incubation in the above test system by serological methods (ELISA), as found by Waldow and Wolf (1997). Wakeham and White (1996) were able to detect resting spores in soil using indirect ELISA or immunofluorescence, with a detection level of 10² spores per g of soil. However, it was found that no serological kit was commercially available at present.

3.4.2 Rhizoctonia solani

The water agar platings of moist sand incubated seed material demonstrated that the pathogen survived this treatment without problems. Incubated samples were also used in biotests in which a sand(3):fine(1):medium(1) vermiculite mixture was used as growth medium (data not presented), and disease development was noted in all pots. However, the growth medium was found unsuited, resulting in highly variable plant emergence, and for that reason no detailed disease scoring was made.

Both in the preliminary and the soil dilution tests it was demonstrated that the pathogen was in high vigour and that the test method was highly sensitive, since even at 0.3 g inoculum per pot a high disease incidence was scored. The 0.3 g inoculum corresponds to 2-4 R. solani colonized wheat seeds, which shows that 2 to 4 inoculum point sources in a pot are sufficient for disease development after three weeks of incubation.

It was verified that the Neogen ELISA kit can be used for identifying R. solani infections in the biotest. Unfortunately the first approach to evaluating whether the ELISA procedure may have a quantitation potential in the present context was unsuccessful, and further, repetitive studies, designed to evaluate this are required, in any event, in order to obtain any safe information on this aspect.

3.4.3 Fusarium oxysporum

As for P. brassicae quite a few research workers have reported serological detection of F. oxysporum by the ELISA method (e.g. Eparvier and Alabouvette, 1994), but no ELISA kit was found to be commercially available.

3.4.4 Tobacco mosaic virus

The tobacco mosaic virus in all three samples incubated in sand clearly survived in amounts that could very easily be detected by the biotests employed. Both tobacco varieties used produced clearly visible symptoms and both reacted strongly positively by ELISA. For the full-scale investigation, use of N. tabacum var. Xanthi-nc alone as test plants should therefore be quite sufficient.

A few of the inoculated leaves showed severe damage (being almost or completely dead) at the time of scoring. This was almost certainly because these leaves had been two young at the time of inoculation. Somewhat older plants should therefore be used for the bioassay, i.e. plants at the 6-8 leaf stage.

3.5 Recommendations for the full-scale investigation

3.5.1 Plasmodiophora brassicae

Since the biotest method as applied here worked well, there are no obvious reasons to modify procedures for the full-scale investigation.

3.5.2 Rhizoctonia solani

Since the most severe disease development was seen in the high temperature regime, this should be used for incubation in the full-scale investigation. The clay-peat growth medium or the sand-clay-peat mixture is suitable for the test. The sowing density should be reduced to 12 seeds per pot.

3.5.3 Fusarium oxysporum

The clay-peat mixture should replace the sand-vermiculite mixture in order to obtain a lower pH-value, which is reported to favour disease development. Bigger pots (2-L) and larger substrate volumes should be used, and the sowing density be reduced to 12 plants per pot, considering the long duration of the test. The temperature regime should be the highest applied in the pilot investigations (25-28?C). Fertiliser should be applied sparingly, and application should be reduced to zero when indications of the first symptoms are recorded. N should be applied as ammonium, not as nitrate, all in order to favour disease development as much as possible and reduce test duration.

3.5.4 Tobacco mosaic virus

It is recommended to use two Nicotiana tabacum var. Xanthi-nc plants for each sample to be tested, inoculating two leaves per plant. The plants have to be at the 6-8 leaf stage for bioassay evaluated by ELISA and counting local lesions.

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