METHOD AND KIT FOR DETECTION THE PRESENCE OF SILVER LEAF DISEASE CHONDROSTEREUM PURPUREUM FUNGUS

Abstract

An antibody that is used to detect the presence of the Chondrostereum purpureum fungus in a plant sample, where the antibody specifically binds to the endolipogalacturonase enzyme produced by Chondrostereum purpureum (anti-endoPG from now). A kit and a method to detect fungal antigens, particularly, to detect the presence of the silverleaf disease (caused by the Chondrostereum purpureum fungus) in fruit trees, by means of detection of the endoPG enzyme through the binding of said antigen to the antibody, functionalized with gold nanoparticles, where the binding is detected via enzyme-linked immunosorbent assays (ELISA) or an immunochromatographic lateral flow assay.

Claims

1. An antibody to detect the presence of the Chondrostereum purpureum fungus in a plant sample, wherein the antibody has a heavy chain defined by the amino acid sequence SEQ ID. 2 and a light chain defined by the amino acid sequence SEQ. ID. 4, and wherein said antibody is conjugated and specifically binds to the enzyme endopolygalacturonase from Chondrostereum purpureum (anti-endoPG antibody).

2. The antibody to detect the presence of the Chondrostereum purpureum fungus in a plant sample according to claim 1, wherein the antibody is conjugated with gold nanoparticles through an L-cysteine residue to form a NpsAu+Lc+Ab complex.

3. The antibody to detect the presence of the Chondrostereum purpureum fungus in a plant sample according to claim 2, wherein the gold nanoparticles have an average diameter of 25 nm and a surface charge of ?19.9 mV.

4. A method for detecting the Chondrostereum purpureum fungus presence in a plant tissue sample comprising the steps of: a. preparing the vegetable or tree tissue sample of interest to obtain a supernatant from the sample, b. establishing contact between the supernatant of the sample to be analyzed with an antibody that specifically binds to the Chondrostereum purpureum endopolygalacturonase enzyme of claim 1, wherein said antibody is in turn conjugated with gold nanoparticles through a L-cysteine residue (NpsAu+Lc+Ab complex), and c. evaluating the specific union of the anti-endoPG antibody, where the sample contains the fungus if the antibody is specifically bound to the sample.

5. The method for detecting the Chondrostereum purpureum fungus presence in a plant tissue sample in accordance with claim 4, wherein, to prepare the sample, 1 g of plant tissue is grinded and crushed, left to macerate, and washed with an organic solvent and the supernatant of the sample is retained.

6. The method for detecting the Chondrostereum purpureum fungus presence in a plant tissue sample in accordance with claim 4, wherein the plant tissue corresponds to leaves, wood, roots, seeds.

7. The method for detecting the Chondrostereum purpureum fungus presence in a plant tissue sample in accordance with claim 4, wherein the specific union of the anti-EndoPG antibody is evaluated using a method selected from the group that consists on a lateral flow immunochromatography or ELISA procedure.

8. A kit for the fast detection of the Chondrostereum purpureum fungus in a plant sample comprising: a reactive strip composed of a nitrocellullose membrane where there are differentiated areas for the sample pad, test zone, control zone and absorption pad, where the test and control zone have the NpsAu+Lc+Ab complex with antibody of claim 1 and the detection secondary antibodies, and instructions of use.

9. The kit for the quick detection of the Chondrostereum purpureum fungus in a plant sample in accordance with claim 8, wherein the detection secondary antibodies correspond to anti-rabbit IgG.

10. The kit for the quick detection of the Chondrostereum purpureum fungus in a plant tissue sample in accordance with claim 8, wherein the sample to be evaluated corresponds to the supernatant of the plant sample

11. The kit for the quick detection of the Chondrostereum purpureum fungus presence in a plant tissue sample in accordance with claim 8, wherein the sample to be evaluated is prepared by grinding and/or crushing plant tissue, then left to macerate, washed with an organic solvent, and the supernatant of this sample is retained.

12. The kit for the quick detection of the Chondrostereum purpureum fungus of claim 8, wherein the plant tissue corresponds to leaves, wood, roots, seeds.

13. Use of the kit for the quick detection of the Chondrostereum purpureum fungus in a plant tissue sample in accordance with claim 8, wherein it is used as a field fungus detection test.

14. The use of the kit for the quick detection of the Chondrostereum purpureum fungus in a plant tissue sample in accordance with claim 8, wherein it is used for in-field fungus detection with a 20-30 minute response time.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0068] The present invention will become more fully understood from the detailed description given hereinbelow and the accompanying drawings which are given by way of illustration only, and thus are not limitative of the present invention.

[0069] FIGS. 1A-1B: UV/VIS spectra of the gold nanoparticles obtained from the S5 synthesis. A) graph shows the optimal nanoparticle wavelength (nm) for its synthesis according to what is described in literature (P. Preechakasedkit et al.,/Biosensors and Bioelectronics(2012) 562-566); b) graph shows wavelength characteristics of the nanoparticles obtained from the S5 synthesis; where A corresponds to the start of the spectrum and B to the end of it. In B), the arrow indicates peak absorbance at 520 nm, equivalent to what is previously described in literature.

[0070] FIG. 2: TEM observation of gold nanoparticles. Nanoparticles obtained from the S5 synthesis are observed. Examination was carried out by transmission electron microscopy (TEM) determining a 25 nm average diameter.

[0071] FIGS. 3A-3B: Size and dispersion distribution determination of the NPsAu. a) The graph shows diameters for the gold nanoparticles obtained from the S5 synthesis, using dynamic light scattering (DLS). b) The graph shows Z potential of the S5 nanoparticles.

[0072] FIGS. 4A-4B: UV-VIS spectra of NPsAu functionalized with L-cysteine. [0073] a) The graph shows the UV/Vis spectra of 200 ?M pure L-cysteine. [0074] b) in the L-cysteine functionalized nanoparticles UV/Vis spectra graph it is possible to observe an increase in functionalized samples (pink, light blue, green, red) in respect to NPs-Au(shown in yellow).

[0075] FIGS. 5A-5B: UV-VIS spectra of functionalized NPsAu conjugated with the antibody. a) the figure shows the UV/Vis spectra of the antibodies. b) the figure shows the UV-VIS spectra of the functionalized nanoparticles that were conjugated to the antibody. In blue, functionalized nanoparticles without antibodies can be observed, while the NPsAu-L-cysteine-antibody complex can be observed in red.

[0076] FIGS. 6A-6B: Chondrostereum purpureum isolation from wood and leaf samples. A) wood sample collection and their conditions are presented, which were used for isolation of the C. purpureum by seeding in aAPD agar solid media. B) collected leaf samples are shown for follow up of disease onset signs and to generate a scale for symptom severity, where 1 corresponds to healthy and 9 to maximum amount of symptoms.

[0077] FIG. 7: Silver leaf detection kit. A) components of the flow device, b) enzyme detection via primary antibody, c) diagram for the interpretation of the kit results, d) validation test on the nitrocellulose membrane, to the left a positive orchard sample and to the right a negative control.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Application Examples

Example 1: Functionalized Gold Nanoparticle Synthesis and Production of the Functionalized Nanoparticle-Immobilized Antibody

[0078] The first step for developing the detection kit comprises gold nanoparticles synthesis.

[0079] Part of the detection kit comprises gold nanoparticles and a reducing agent, producing a stable colloidal solution. For that purpose, monodisperse colloidal gold was synthetized using the modified Turkevich method. 6 different tetrachloroauric acid and trisodium citrate aqueous solutions were prepared, and different HAuCl.sub.4 and trisodium citrate oncentrations were evaluated to select the solution that met the desired characteristics for a gold nanoparticle (NPsAu) solution (Table 1).

TABLE-US-00001 TABLE 1 Solutions used in gold nanoparticles synthesis. Nanopure 25 mM 1% Sodium water HAuCl.sub.4 citrate Synthesis (mL) (mL) (mL) Observations S1: 100 1 2.5 Temperature of 95? C., citrate is added, 20 min agitation. Orange color. S2 20 1 2 Temperature of 95? C., citrate is added, 20 min agitation. Orange color. S3 100 0.1 2 Temperature of 95? C., citrate is added, 20 min agitation. Brilliant red color. S4 100 0.3 2 Temperature of 95? C., citrate is added, 20 min agitation. Red color, black residues. S5: 100 0.2 2 Temperature of 95? C., citrate is added, 20 min agitation. Red color. S6: 100 0.2 4 Temperature of 95? C., citrate is added, 20 min agitation. Red color, citrate excess.

[0080] Solution characteristics were determined, according to their colorimetric properties, bright red color and a visible light spectrum absorbance (UV-VIS) wavelength peak at 520 nm. From all possible combinations from table 1, solution 5 (S5) was selected for NPsAu synthesis, because it showed Kiger colloidal stability, a bright red color, both characteristics for the absorbances reported in literature P. Preechakasedkit et al.,/Biosensors and Bioelectronics(2012) 562-566 (FIGS. 1A-1B). In any case, pre combinations were tested by modifying the citrate concentration, however, these combinations did not reveal the desired red color, so they were discarded.

[0081] On the other hand, the NPsAu obtained through S5 synthesis were characterized with high resolution transmission electron microscopy TEM), where it was possible to observe monodispersed spherical particles with an average diameter of 25 nm (FIG. 2). Electric charge or Z potential of the nanoparticles, average size and s distribution were determined with the dynamic light scattering (DLS) method using the Malvern Zetasizer nano ZS equipment (FIG. 3A; Table 2). Results for these measurements showed that the superficial charge of the NPsAu was ?19.9 mV, which is explained by the negative charges that are conferred by citrate, explaining the proper nanoparticle dispersion obtained, which presented a polydispersity index lower than 0.3 (FIG. 3B; Table 3).

TABLE-US-00002 TABLE 2 Size-distribution by S5 NPsAu intensity. Analysis conditions Results Temperature ? C. 25.1 Z-average (d .Math. nm) 23.07 Count rate (kcps) 293.7 PdI 0.219 Measuring position 4.65 Intercept 0.897 Size 27.22 Intensity % 100.00 Standard deviation 9.885

TABLE-US-00003 TABLE 3 Zeta potential distribution (superficial charge) of S5 NPsAu. Analysis conditions Results Temperature (? C.) 25.1 Zeta Potential (mV) ?19.9 Count rate (kcps) 97.1 Zeta Deviation (mV) 50.4 Measuring position (mm) 2.00 Conductivity (mS/cm) 0.290 Zeta runs 10

[0082] The second step corresponds to the nanoparticle functionalization, with the objective of obtaining NPsAu that can be used as diagnostic agents. Nanoparticle (NPsAu) surfaces were adapted for further conjugation with a ligand or molecule that can be used to control nanoparticle size during its synthesis and to prevent their agglomeration. In this case, the molecules used to conjugate to NPsAu correspond to antibodies, that were linked through a L-cysteine residue to the NPsAu because said residue has a carboxyl moiety that can help with the stabilization of the conjugation.

[0083] For nanoparticle functionalization, NPsAu were synthetized using the Turkevitch method, and then they were conjugated with L-cysteine starting from a 200 ?M stock solution and performing dilutions (0.1; 0.2; 0.4; 1.2; 4 ?M). This was performed with the objective of determining optimal liquid ligand concentration that allows to maintain particle size without them showing signs of agglomeration. Prepared solutions were stirred for 2 hours at room temperature and then were centrifuged at 10000 rpm for 15 minutes. Finally, NPsAu-cysteine UV-VIS spectra was verified using as a control the UV-VIS absorbance spectra of pure L-cysteine, where 200 ?M of the latter were used (FIG. 4A). L-cysteine functionalized nanoparticles show an absorbance increase in functionalized nanoparticles (shown in pink, light blue, green, red) in respect to NPsAu (yellow) (FIG. 4B).

[0084] Endo Antibodies

[0085] The EndoPG antibody that is part of the NpsAu+Lc+Ab complex described on the present invention is comprised by a heavy chain and a light chain. The heavy chain of said antibody is comprised by a heavy chain that presents a nucleotidic sequence represented by the SEQ ID. 1 and an aminoacid sequence represented by the SEQ ID. 2

[0086] On its part, the light chain of said antibody has a nucleotidic sequence represented by SEQ ID. 3 and an aminoacid sequence represented by SEQ ID. 4.

[0087] The antibody can be synthetized through any process that allows for synthetic antibody production. It is known that it is possible to synthesize an antibody when its nucleotidic and aminoacidic sequences are defined.

[0088] Its chemical synthesis is possible through molecular methods for transformation of microorganisms or host organisms that produce the antibody, methods that rely on recombinant bacterial, animal, human cells or hybridomas for production of antibodies. A variety of different host organisms are considered, including E. coli, Saccharomyces cerevisiae, Pichia pastoris, cells from insects, algae, plants, and cells from mammals. Cell-free systems are included as methods for generating genomic libraries, with a cell-free production denomination.

[0089] Antibody Immobilization on Functionalized NPsAu

[0090] For antibody immobilization in functionalized NPsAu nanoparticles, an antibody solution was prepared by adding 1 mL of sterile water to lyophilized antibodies. Then, a 1:1000 dilution was performed (10 ?L of antibody with 990 ?L of sterile water) and was used in subsequent assays. Moreover, a wash solution consisting in 1 mL of 10?PBS with 100 ?L of 10% BSA and 9 mL of sterile water was prepared.

[0091] 1 mL of L-cysteine mixed with 8 ?L of antibody dilution were added to 5 1.5 mL tubes and then were incubated for 30 minutes at room temperature. Then, 100 ?L of 10% BSA were added to the tubes and were incubated for 10 minutes at room temperature. Subsequently, tubes were centrifuged at 20000 g for 1 hour at 4? C. The supernatant was discarded and remaining precipitate was resuspended in 1 mL of wash solution and centrifuged; the obtained pellet was resuspended in water and adjusted pH to 7.0.

[0092] Absorbance peak movement from 520 to 530 nm and a lower band intensity (less absorbance) were observed using UV/VIS absorbance (FIG. 5B). Latter results, together with the red coloring developed by the solution, indicate that the antibody immobilization on the nanoparticle was successful. It is worth mentioning that absorbance for the unconjugated antibody was also verified (FIG. 5A)

Example 2: Anti-endoPG Specificity Validated in Different Fruit Plants

[0093] To determine antibody specificity in different fruit trees, the proposed ELISA protocol was performed to detect endoPG1 produced by C. pupureum, in apple, blueberry, plum, cherry, peach and kiwi. To validate ELISA standardization in different fruit trees, samples were gathered from commercial orchards that were already infected with silver leaf (monthly gathering from October 2017 to May 2018) (Table 4.)

TABLE-US-00004 TABLE 4 Silver leaf-contaminated plant sample gathering areas for evaluation of the functionalized NPsAu specificity for endoPG. Host Gathering area Apple Santa Rosa, Chill?n, B?ob?o region. Blueberry Vilc?n, Temuco, Biob?o, Maule. Kiwi Marchant Island, Curic?, Maule. Cherry Los Guindos, Curic?, Maule. Plum Tutuquen, Curic?, Maule. Peach Pichidegua, Cahapoal, O'Higgins.

[0094] In each of the gathering areas indicated on table 4, wood samples were collected from plants that showed foliar symptoms to isolate the pathogen. Pathogen isolation was performed by depositing samples on 25% acidified potato dextrose agar (25% aAPD, Difco), and then incubating in a moist chamber for visible signs of silver leaf disease (FIG. 6A). Besides, leaves were gathered from a 1.2 m height from ground level to be stored at ?20? C. and analyzed afterwards.

[0095] In the first instance, a scale for symptom severity determination was used when examining leaves, where said scale goes from 1 to 9 and where 1 corresponds to a healthy specimen and increases as the disease does to 9 (FIG. 6B). The total number of samples that were gathered for analysis was of 430 in a 30-day period, analyzing foliar symptoms of each one of them as it is indicated in FIG. 6B.

[0096] A chromatographic analysis of the endopolygalacturonase (endoPG) enzyme produced by the fungus responsible for the foliar symptoms of the silver-leaf disease was performed on these samples (Senda et al., 2001).

[0097] The total number of samples that were analyzed corresponds to 24 per species, considering 3 biological replicates (pertaining to 3 different plant leaves) and 2 technical replicates (grinding supernatant of the same biological replicate). The analysis was performed using the ELISA technique, where leaves were subjected to grinding and crushing on buffer, then were incubated with primary and secondary antibody, to then determine the OD at a 450 nm wavelength to determine endoPG concentration (having already performed a calibration curve using synthetic peptides).

[0098] On the other hand, nursery plans were used as control, which were inoculated with virulent strains of the pathogen, checking presence of the pathogen by isolating in culture media and detecting with PCR, just as was checked on plants naturally inoculated with the pathogen. The antibody was able to detect the enzyme in each one of the cases, excepting kiwi, that underwent some modification on its detection protocol. Plants were considered to be sick if showed at least twice the endoPG concentration (Herrera et al., 1995), in respect to the average value of 10 negative controls. Treatments that were used and detected concentrations are presented next (Table 5):

TABLE-US-00005 TABLE 5 Treatments used to verify differences on endoPG concentration present in plants with different silver leaf symptom intensities. EndoPG Treatment Sample concentration/gpf Symptomatology 1 48 0.0007317 Symptomatic 2 25 0.0005834 Symptomatic 3 73 0.0005421 Symptomatic 4 41 0.0005369 Asymptomatic 5 40 0.0004867 Asymptomatic 6 15 0.0004187 Asymptomatic 7 37 0.0004122 Asymptomatic 8 3 0.0004024 Asymptomatic 9 39 0.0004019 Asymptomatic 10 17 0.0003668 Asymptomatic 11 36 0.0003623 Asymptomatic 12 18 0.0003614 Asymptomatic 14 38 0.0000974 Asymptomatic 15 100 0.0000754 Asymptomatic 16 99 0.0000701 Asymptomatic 17 C2 0.0001477 Healthy control 18 C2 0.0001406 Healthy control

[0099] Results show that, for plum, cherry, peach and blueberry, minimum detection levels in asymptomatic plants were considerably higher than twice the control values. For apple, on the other hand, was considerably higher in each case, but in some of them, no superior than twice the control values.

Example 3: Kit Preparation for Early Detection of Silver Leaf in Fruit Orchards: Functionalized Nanoparticle Sensibility Determination in Reactive Strips

[0100] To determine if the nanoparticles are capable of detecting endoPG, an evaluation assay was performed in nitrocellulose membrane strips with nanoparticles on them, where one of their ends was submerged in the grinding supernatant of apple foliar samples from plants infected with the pathogen. The supernatant moved along the nitrocellulose strip by capillary diffusion unto the nanoparticles, showing a circumference on the strips where the latter were added.

[0101] Conjugate specificity was determined by means of the interaction of enzyme preparations that show endoPG1-like functions with it, using an in-solution colorimetric assay. The assay was performed by testing two commercial enzymatic preparations solutions composed by a mixture of enzymes, corresponding to the Natuzym DP Ultra product, comprised of pectolyase, polygalacturonase, pectinesterease and arbanase, and DeltaZym VR AC-100 with cellulases and hemicellulases. Both enzymatic preparations were in a 1:10 and 1:100 ratio with water and PBS buffer respectively. The reaction was performed by adding 6 mL of the enzymatic complex to 200 ?L of nanoparticles. Results of this assay showed that no color change occurred in any of the reactions in respect to the 1:10 control. An UV-VIS spectrophotometric analysis delivered curves for each of the different reactions and it showed that they followed the same absorbance pattern, indicating that the antibody did not recognize any of those enzymes.

Example 4: Immunoassay Kit and Evaluation of its Detection

[0102] The immunoassay kit was prototyped based on the Tucker et al., 1980; Nogata et al., 1993; Priya et al., 199, and Srivastava y Dwivedi, 2000, studies.

[0103] The immunoassay kit is comprised by a nitrocellullose membrane reactive strip composed by four areas or zones, which correspond to the sample pad, the test zone, the control zone and an absorption pad placement (FIG. 7, panel A). The nanoparticle complex is applied on the lower part of the membrane. The test zone and control zone both contain primary and secondary antibodies and then the adsorption pad is placed.

[0104] The detection is based on the diffusion of conjugated gold nanoparticles through the nitrocellulose membrane, that by means of capillary diffusion move towards specific adsorption sites for enzyme detection by primary and secondary antibodies that are immobilized onto it. The reaction used a synthetic endoPG1, which reacted with the lines that contained NpsAu+Lc+Ab complex on the nitrocellulose strip and with the control line. Protocols were optimized for a reaction time of 15 to 20 minutes (FIG. 7, panel B). Specifically, proper functioning of the kit comprises diffusion of the NPsAu where the NpsAu+Lc+Ab complex is rehydrated and is mixed with the sample. When the sample contains endoPG, the enzyme is captured by the antibody and this new complex diffuses towards the test area, where its retained by the membrane immobilized anti-endoPG antibody, detectable by a red colored line. Anti-endoPG/Np complex excess that surpasses the test area keeps on diffusing to the control area where it reacts with the anti-rabbit IgG secondary antibody, developing a red color (control), validating the method of detection (FIG. 7, panel A). If a red color appears on both the control and test zones the sample is considered to be positive. If the kit does not detect endoPG on the sample, only one red colored area will appear in the control zone, because immobilization of the anti endoPG antibody/Nps complex does not occur due to absence of the antigen (FIG. 7, panel D).

Example 5: Kit Concept Test in 6 Fruit Orchards of Economic Interest

[0105] To start the kit concept test an enzymatic extract was prepared and added to the nitrocellulose membrane that has the immobilized antibodies. This extract was prepared by weighing 1 g of sample and cutting in small pieces by hand. Then, it was macerated with 3 mL of 1?PBS, the mash was retained and then washed with acetone. Remaining residue was resuspended in 1?PBS buffer and stirred. Finally, the pellet was discarded and the supernatant was retained, the last one corresponding to the enzymatic extract that reacts with the components on the reactive strip.

[0106] The prototype was validated in the laboratory using blueberry samples from. Vilc?n, and apple tree samples from Chill?n, naturally infected with C. purpureum, and also artificially inoculated plants with virulent strains of the fungus. For the negative control, in vitro plant leaves were used.

[0107] In both species an approximate reaction time of 20 min was observed.

[0108] The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

BIBLIOGRAPHY

[0109] INIA, Instituto de investigaciones agropecuarias. (2018). Investigadores INIA presentaron investigaci?n en plateado de los frutales en Congreso de Fitopatolog?a en EEUU. https://www.inia.cl/blog/2018/09/13/investigadores-inia-presentaron-investigacion-en-plateado-de-los-frutales-en-congreso-de-fitopatologia-en-eeuu/ [0110] INIA. (2006). Revista Tierra Adentro: frutales y vi?as. http://biblioteca.inia.cl/medios/biblioteca/ta/NR33449.pdf [0111] P. Preechakasedkit et al.,/Biosensors and Bioelectronics(2012) 562-566. [0112] Portal frut?cola (2019). Enfermedad del plateado en carozos y ard?danos. https://www.portalfruticola.com/noticias/2019/08/07/enfermedad-del-plateado-en-carozos-y-arandanos/30 [0113] Rojo C, Becerra V, France A, Paredes M, Buddie A, alzarini M. (2017). Genetic diversity of Chondrostereum purpureum (Pers.) Pouzar causing silverleaf disease on blueberries in Chile. Gayana Bot. 74(1): 176-188. [0114] France, A., Grinbergs, D. & Carrasco, J. (2016). First detection of silverleaf (Chondrostereum purpureum) on rabbiteye blueberry (Vaccinium virgatum) and disease damages. XI International Vaccinium Symposium 1180. [0115] Grinbergs, Daina & Chilian, J. & Lisboa, K. & France, Andres. (2019). First Report of Silverleaf Disease Caused by Chondrostereum purpureum on Murta (Ugni molinae) in Chile. Plant Disease. 103. 2140-2140. 10.1094/PDIS-12-1bakan,8-2285-PDN. [0116] Grinbergs, D.; Chilian, J.; Carrasco-Fernandez, J.; France, A.; Moya-Elizondo, E.; Gerding, M. A PCR-based method for the rapid detection of Chondrostereum purpureum in apple. Plant Dis. 104, 71, 702-707. [0117] Grinbergs, D.; Chilian, J.; Hahn, C.; Reyes, M.; Isla, M.; France, A.; B?rve, J. Silverleaf (Chondrostereum purpureum) Effects on Japanese Plum (Prunus salicina). Preprints.org 2021, 2021060160. https://doi.org/10.20944/preprints202106.0160.v1