Soil testing diagnosis

DNA extraction method

5.3.2.1.

The two DNA extraction methods were shown to successfully extract DNA from the fungal propagules since a portion of the DNA that encoded the rRNA genes encompassing the ITS and 5.8S regions was amplified from the DNA extracts.

As the DNA extracts may have contained impurities that inhibited PCR different volumes (2, 1, 0.5 and 0.2 μL) were tested to determine the minimum volume that could produce consistent amplification. For Method 1 (Figure 5.1 A), PCR products of approximately 550 and 600 bp were visualised on a gel for all samples except for 2 µL of DNA with a standing time of 6 min and for 1 µL with a standing time of 3 min. For Method 2 (Figure 5.1 B), faint bands were present for all DNA samples using 0.2 µL of DNA and for 103 conidia /g of soil using 1 µL of DNA whereas bright bands were only observed for 105 conidia /g of soil using 1 µL of DNA. The negative controls for both methods did not amplify any products and the positive control which consisted of 1 µL of DNA from isolate M1 showed a band at 550 bp.

From these results, Method 2 was discarded and Method 1 was used for the following DNA extraction from soil.

Figure 5.1. PCR products obtained with the universal fungal primers ITS4 and ITS1F, using 2, 1, 0.5 and 0.2 µL of DNA extract from 10 g of soil mixed with 0, 10, 103 and 105 C. destructans conidia /g using two DNA extraction methods (- : negative control; + positive control). A: Method 1 using soil without Cylindrocarpon spores (standing times of a: 3 min, b: 6 min and c: 10 min). B: method 2 using the four quantities of spores.1kb – 1 kb plus DNA Ladder (Invitrogen).

Nested species specific PCR

5.3.2.2.

When 0.2 µL of DNA was selected for use in the first PCR of the nested PCR, it produced bands of similar brightness for all standing times compared to the 0.5 and 1.0 µL quantities and therefore was the least likely to inhibit the PCR.

The nested PCR using species specific primers for C. macrodidymum (Figure 5.2 A) amplified a 300 bp product for DNA extracted from soil containing 105 conidia /g of soil with all three standing times. For C. liriodendri (Figure 5.2 B), a 200 bp band was amplified for DNA extracted from soil containing 105 conidia /g of soil with standing times of 6 min and 10 min. For C. destructans (Figure 5.2 C), PCR products were observed on a gel for DNA extracted from soil containing 103 and 105 conidia /g of soil with standing times of 6 and 10 min. However, a 200 bp band was also present for soil to which Cylindrocarpon conidia had not been added. It was very faint with a standing time of 6 min and very strong with a 10 min

1kb 0 10 103 105 0 10 103 105 0 10 103 105 0 10 103 105 - + 2 μL 1 μL 0.5 μL 0.2 μL B 1kb a b c a b c a b c a b c - + 2 μL 1 μL 0.5 μL 0.2 μL A

resident population of C. destructans. None of the negative controls showed a band and all the positive controls amplified a product of the appropriate size. The sequencing result showed that the primers were specific to the different species.

Figure 5.2. PCR products obtained with species specific primers in a nested PCR of DNA extracted from soil mixed with 0, 10, 103 and 105 Cylindrocarpon conidia /g of soil using DNA extraction Method 1 (- : negative control; + positive control; standing times of a: 3 min, b: 6 min and c: 10 min). A: C. macrodidymum. B: C. liriodendri. C: C. destructans. 1kb – 1 kb plus DNA Ladder (Invitrogen).

The pathogens were detected in soil containing conidia at a concentration of 1 × 105 conidia /g of soil. To determine the detection sensitivity of the nested PCR in terms of pg DNA and conidia the following calculations were applied:

1kb 0 0 0 10 10 10 103 103 103 105 105 105 - - + a b c a b c a b c a b c 100 bp - 500 bp - A B 1kb 0 0 0 10 10 10 103 103 103 105 105 105 - - + a b c a b c a b c a b c 100 bp - 500 bp - 1kb 0 0 0 10 10 10 103 103 103 105 105 105 - - + a b c a b c a b c a b c C 100 bp - 500 bp -

To extract DNA using Method 1, 10 g of soil containing 106 conidia was initially suspended in autoclaved distilled water with agar and 1/3 of the pellet was used for DNA extraction. Thus, DNA from approximately 3.3 × 105 conidia was extracted and resuspended in 100 µL:

Number of conidia from which DNA was extracted (Y) = (X conidia /10 g soil) × 10 g /3

As only 0.2 µL (1/500th) of the resultant DNA extract was used for the first PCR and only the 106 concentration produced an amplimer for C. macrodidymum and C. liriodendri, the DNA from approximately 6.7 × 102 conidia was amplified.

Actual number of conidia whose DNA was amplified by nested PCR = Y/500 (where Y = the number of conidia from which DNA was extracted)

If one Cylindrocarpon haploid genome is considered to have approximately the same weight as Fusarium oxysporum (both fungi belong to the Nectriaceae family) with 0.0377 pg (Pasquali et al., 2006) and one conidium has approximately 3 nuclei, the DNA in one conidium would weigh 0.11 pg.

DNA in one conidium = 0.0377 pg /nucleus × 3 nuclei /conidium

As 6.7 × 102 conidia were able to be detected from each of the different species, the resolution was equivalent to 75.4 pg of DNA.

DNA (pg) amplified = 6.7 × 102 conidia × 0.11 pg DNA.conidium-1

670 conidia or 75.4 pg of DNA were capable of being detected by nested PCR

For soil testing diagnostics using nested PCR, DNA extraction Method 1 with a standing time of 10 min, 0.2 µL of the DNA extracted in a primary PCR using general tubulin primers and the diluted products (1:100) for the secondary PCR using species specific primers was selected.

Quantitative PCR

5.3.2.3.

5.3.2.3.1. DNA extraction from cultures

Quantitative PCR of DNA extracted from a pure culture of isolate L1 using species specific primers Cyli F1 and Cyli R1 produced an amplicon using the standard PCR thermal cycle described in Section 3.2.3.2 at template concentrations of 30 ng, 3 ng, 0.3 ng, 30 pg and 3 pg (Figure 5.7). Gel electrophoresis of the amplicons showed that they were of predicted size (200 bp). All threshold cycle values (Ct) above 39 were discarded as described in Section 3.3.4. The standard curve showed a linear correlation between the logarithm of the concentration and the threshold cycle (Ct) values with a correlation coefficient (r2) of 0.992. The regression equation of the standard curve was: Ct= -4.03 (log [DNA]) + 29.67.

The detection limit of the qPCR was calculated for a Ct value of 39: log [DNA ng] = (39 - 29.67) /-4.03

= 4.8 pg of genomic DNA

Quantitative PCR of DNA extracted from a pure culture of isolate M3 using species specific primers Cyma F1 and Cyma R1 produced an amplicon which was of predicted size (300 bp) for 30 ng, 3 ng, 0.3 ng and 30 pg of template DNA (Figure 5.8). The standard curve showed a linear correlation between the logarithm of the concentration and the threshold cycle (Ct) values with r2 of 0.997. The regression equation of the standard curve was: Ct= -4.29 (log [DNA]) + 30.69. The detection limit of the qPCR was 11.5 pg of genomic DNA.

Quantitative PCR of DNA extracted from a pure culture of isolate D2 using species specific primers Cyde F1 small and Cyde R2 produced an amplicon which was of predicted size (200 bp). The standard curve showed a linear correlation between the logarithm of the concentration and the threshold cycle (Ct) values, with r2 of 0.945. The regression equation of the standard curve was: Ct= -5.00 (log [DNA]) + 33.1. The detection limit of the qPCR was 66 pg of genomic DNA.

5.3.2.3.2. DNA extraction from conidia

For C. liriodendri, results from the qPCR indicated an average amount of 321 pg of DNA for 104 conidia, 22.1 pg of DNA for 103 conidia and 5.7 pg of DNA for 102 conidia. No amplicons were detected for 10 conidia and the negative control. When these results were graphed using Excel version 2003, the fitted line showed a linear correlation between the logarithm of the number of conidia and the logarithm of the quantity of DNA, with a correlation coefficient (r2) of 0.972 (Figure 5.3). The equation of the curve was:

One C. liriodendri conidium is equivalent to 0.14 pg of DNA.

If one Cylindrocarpon haploid genome is considered to have approximately the same weight as F. oxysporum with 0.0377 pg and one conidium is equivalent to 0.14 pg of DNA, one C. liriodendri conidium is constituted of approximately 4 nuclei.

As the theoretical detection limit for C. liriodendri is 4.8 pg of DNA (Section 5.3.2.3.1) and one conidium contains 0.14 pg of DNA, 4.8 pg of DNA is equivalent to 34.3 conidia. If 34.3 conidia can be detected in 1 μL, then 3430 conidia can be detected in 100 μL. As DNA was extracted from a third of 10 g of soil, approximately 1029 conidia can be detected in 1 g of soil. Therefore, the theoretical detection limit for C. liriodendri is 1029 conidia /g of soil (1.0 x 103 conidia/g soil).

The products obtained with qPCR were confirmed on a 1.5 % agarose gel. A band at 200 bp was present when DNA extracted from 102, 103 and 104 C. liriodendri conidia was amplified (data not shown).

Figure 5.3. Fitted line showing the correlation between number of conidia from which DNA was extracted and DNA quantity estimated by the genomic DNA standard curve for C. liriodendri.

For C. macrodidymum, results from the qPCR indicated an average amount of 438.5 pg of DNA for 104 conidia, 109.5 pg of DNA for 103 conidia and 13.3 pg of DNA for 102 conidia. No amplicons were detected for 10 conidia and the negative control. When these results were

y = 0.8x - 0.85 r2 = 0.9726 0 1 2 3 1 2 3 4 log (conidia) lo g (D N A q u a n ti ty )

logarithm of the number of conidia and the logarithm of the quantity of DNA, with a correlation coefficient (r2) of 0.983 (Figure 5.4). The equation of the curve was:

log [DNA quantity] = 0.87 log [conidia number] – 0.75.

This equation indicates that DNA quantity = 10-0.75 for one conidium. One conidium is equivalent to 0.18 pg of DNA.

If one Cylindrocarpon haploid genome is considered to have approximately the same weight as F. oxysporum with 0.0377 pg and one conidium is equivalent to 0.18 pg of DNA, one C. macrodidymum conidium is constituted of approximately 5 nuclei.

Figure 5.4. Fitted line showing the correlation between number of conidia from which DNA was extracted and DNA quantity estimated by the genomic DNA standard curve for C. macrodidymum.

As the theoretical detection limit for C. macrodidymum is 11.5 pg of DNA (Section 5.3.2.3.1) and one conidium weighs 0.18 pg of DNA, 11.5 pg of DNA is equivalent to 63.9 conidia. If 63.9 conidia can be detected in 1 μL, then 6390 conidia can be detected in 100 μL. As DNA was extracted from 10/3 g of soil, approximately 1917 conidia can be detected in 1 g of soil. Therefore, the theoretical detection limit for C. macrodidymum is 1917 conidia /g of soil (1.9 x 103 conidia /g soil).

The products obtained with qPCR were confirmed on a 1.5 % agarose gel (Figure 5.5). A band at 300 bp was present when DNA extracted from 102, 103 and 104 C. macrodidymum conidia was amplified.

y = 0.87x - 0.75 r2 = 0.9836 0 1 2 3 1 2 3 4 log (conidia) lo g (D N A q u a n ti ty )

Figure 5.5. PCR products obtained with species specific primers Cyma F1 /Cyma R1 in a qPCR of different DNA quantities extracted from pure culture of C.

macrodidymum (30 ng to 3 pg) and of DNA extracted from 0.1, 1, 10, 102, 103 and 104C. macrodidymum conidia(- : negative control). 1kb – 1 kb plus DNA Ladder (Invitrogen).

For C. destructans, further optimisation of the qPCR was required as the detection limit obtain for this species with pure cultures was 66 pg (Section 5.3.2.3.1) and this limit was likely to increase when the PCR was used with propagules in soil. Therefore, amplification of conidia from this species was not tested.

5.3.2.3.3. DNA extraction from soil over time

DNA from the different soil samples was extracted using a modification of the optimised Method 1. Ten grams of soil were mixed with 45 mL (instead of 90 mL) of sterile water that contained 0.01% of agar and left to stand for 10 min. The agarose gel in Figure 5.6 shows the DNA extracted from the different samples. The amount of DNA extracted from the soil sample taken at time 0 (immediately after inoculation) is higher than any extracted in the following weeks.

Figure 5.6. Visualisation on 1 % agarose gel of DNA extracted from soil infected with the three Cylindrocarpon species and the controls after 0,1,2,3 and 6 weeks (C: control; D: C. destructans; M: C. macrodidymum; L: C. liriodendri; 0 to 6: weeks after soil inoculation). 1kb – 1 kb plus DNA Ladder (Invitrogen).

1kb 30 3 0.3 30 3 - 104 103 102 10 1 0.1 ng ng ng pg pg 200 bp 500 bp 1kb C0 C1 C2 C3 C6 D0 D1 D2 D3 D6 L0 L1 L2 L3 L6 M0 M1 M2 M3 M6 - 500 pb - 200 pb

At time 0, 10 g of soil containing 106 conidia was initially suspended in autoclaved distilled water with agar and 1/3 of the pellet was used for DNA extraction, thus DNA from approximately 3.3 × 105 conidia was extracted and suspended in 100 µL. As 1 µL was used for the nested PCR, the DNA from approximately 3.3 × 103 conidia was available for amplification.

For C. liriodendri, results from the qPCR indicated an average amount of 57.1 pg of pathogen DNA was detected in the 1 μL of DNA extracted from the soil samples infested with 106C. liriodendri conidia /g of soil at time 0.

As the fitted line in Section 5.3.2.3.2 for C. liriodendri conidia indicated that log [conidia number] = (log [DNA quantity] + 0.85) /0.8,

log [conidia number] = (log [57.1]+ 0.85) /0.8 57.1 pg is equivalent to 1.8 × 103 conidia

As 1 μL of DNA extracted from the 100 μL of DNA extracted was used for the qPCR, 1.8 × 103 conidia × 100 μL DNA extraction = 1.8 × 105 conidia were present in 3.3 g of soil. In 1 g of soil, 1.8 × 105 conidia /3.3 g of soil = 5.5 × 104 conidia were actually detected.

As 1 × 105 conidia /g of soil were initially mixed with the soil, 1.8 times less conidia were detected by qPCR at time 0.

Agarose gel electrophoresis of qPCR amplimers showed that a faint band was obtained for soil samples from 1, 2 and 3 weeks after infestation and these corresponded to 3.9, 3.0 and 3.2 pg of DNA, respectively (equivalent to 64, 46 and 50 conidia using the fitted line in Section 5.3.2.3.2) in 1 μL of DNA extraction. These results are equivalent to 1.9 × 103, 1.4 × 103 and 1.5 × 103 conidia /g, which represent 3.5, 2.5 and 2.7% of the initial conidium number, respectively when considering 5.5 × 104 conidia at time 0.

No amplicons were detected for the different controls or from the inoculated soil at week 6. The molecular weight of the products obtained with the qPCR was confirmed as the correct size on a 1.5 % agarose gel (Figure 5.7), and the sequencing result showed that the primers were specific to the different species.

Figure 5.7. PCR products obtained with species specific primers Cyli F1 /Cyli R1 in a qPCR of different DNA quantities extracted from pure culture of C. liriodendri and of DNA extracted from soil mixed with 106 C. liriodendri conidia after 0, 1, 2, 3 and 6 weeks (- : negative control; L: C. liriodendri; C: control; 0 to 6: weeks after soil inoculation). 1kb – 1 kb plus DNA Ladder (Invitrogen).

For C. macrodidymum, results from the qPCR indicated an average amount of 115.5 pg (equivalent to 1.7 × 103 conidia using the fitted line in Section 5.3.2.3.2 for C. macrodidymum conidia) of pathogen DNA was detected in 1 μL of DNA extracted from the soil samples infested with 106C. macrodidymum conidia /g of soil at time 0.

As the fitted line in Section 5.3.2.2 for C. macrodidymum conidia indicated that [DNA quantity] = 0.87 log [conidia number] – 0.75,

log [conidia number] = (log [115.5]+ 0.75) /0.87 115.5 pg is equivalent to 1.7 × 103 conidia

As 1 μL of DNA extracted from the 100 μL of DNA extracted was used for the qPCR, 1.7 × 103 conidia × 100 μL DNA extraction = 1.7 × 105 conidia were present in 3.3 g of soil. In 1 g of soil, 1.7 × 105 conidia /3.3 g of soil = 5.2 × 104 conidia were actually detected. As 1 × 105 conidia /g of soil were mixed with the soil, 1.9 times less conidia were detected.

No amplicons were detected for infested soil samples or the controls 1, 2, 3 or 6 weeks after infestation. The molecular weight of the products obtained with the qPCR was confirmed as the correct size on a 1.5 % agarose gel (Figure 5.8). A single band at 300 bp was present when the different DNA concentrations from pure culture of C. macrodidymum and the soil sample from C. macrodidymum at time 0 were amplified. No band was visualised in the controls.

500 pb

200 pb

1kb 30 3 0.3 30 3 - L0 L1 L2 L3 L6 C0 C1 C2 C3 C6 1kb

Figure 5.8. PCR products obtained with species specific primers Cyma F1 /Cyma R1 in a qPCR of different DNA quantities extracted from pure culture of C.

macrodidymum and of DNA extracted from soil mixed with 106 C.

macrodidymum conidia after 0, 1, 2, 3 and 6 weeks (- : negative control; M: C. macrodidymum; C: control; 0 to 6: weeks after soil inoculation). 1kb – 1 kb plus DNA Ladder (Invitrogen).

5.4. Discussion

The objective of this chapter was to develop a soil testing method for the detection of Cylindrocarpon spp. Different baits were used to isolate the pathogen, however, none were successful with natural soil. Cylindrocarpon destructans was successfully isolated from apples inoculated with autoclaved potting mix infested with 5 x 104 conidia for both apple varieties and 5 x 102 conidia for apple variety Granny Smith. Mwanza and Kellas (1987) isolated C. destructans from soil using this method, however, the main fungal species isolated were Pythium, Penicillium and Fusarium as observed in this experiment. Sutherland et al. (1966) isolated Pythium vexans and Trichoderma lignorum using the apple baiting technique. In this soil study, the pathogen may have been masked by the fast growing fungi found on the PDA plates, may not have been able to compete with the microorganisms present in the soil or may have rapidly converted into chlamydospores which did not germinate and invade the apple tissue. In contrast, when placed in autoclaved potting mix the pathogen does not compete with other microorganisms and can multiply in the environment as observed by Taylor (1964) who reported that Cylindrocarpon species in contact with sterile soil develop hyphae while in non autoclaved soil, the pathogen produced chlamydospores. As chlamydospores need to be stimulated by the environment to germinate (Matturi and Stenton, 1964), the infection process would be expected to be slow. As a result, this method was discarded.

1kb 30 3 0.3 30 3 - M0 M1 M2 M3 M6 C0 C1 C2 C3 C6 1kb ng ng ng pg pg

200 bp- 500 bp-

An alternative to the apple bait method was to use seedlings of different plant species as bait. Cylindrocarpon species are known to have a wide host range (Booth, 1966; Brayford, 1992) and C. destructans has been recovered from roots of clover (Skipp et al., 1986), dwarf bean (Taylor and Parkinson, 1965), pea (Matturi and Stenton, 1964), carrot (Sweetingham, 1983) and parsnip (Channon and Thomson, 1981; Sweetingham, 1983). These plants were selected for their availability, their low cost, the time required for their germination and their susceptibility to Cylindrocarpon spp. For spinach and pea seedlings sown into autoclaved potting mix infested with C. destructans conidia, the damping off symptoms that developed contained the pathogen, however, when the experiment was repeated with infested garden soil the pathogen was not recovered from the seedlings. Similar observations were reported by Sweetingham (1983) who investigated the pathogenicity of Cylindrocarpon species towards Pinus radiata seedlings. In autoclaved media, the pathogen caused damping off or root stunting of the pine seedlings while in non sterile media inoculated with C. destructans, none of the seedlings were affected (Sweetingham, 1983). The pathogen was clearly not able to infect the seedlings in soil with other microorganisms present.

Since Parkinson and Thomas (1969) reported that the frequency with which “C. radicicola” (C. destructans) was isolated from dwarf bean root surfaces increased with time (with an incidence of 4.5 and 35.6% after 1 and 6 months, respectively), an experiment that used a longer period of growth might be needed for the pathogen to infect or colonise seedling roots. In Chapter 4, the pathogen was recovered from grapevine stems 6 months after the soil was inoculated with different Cylindrocarpon propagules. However, this was not explored as a