ES2817831T3 - Method of diagnosis and treatment - Google Patents

Abstract

Method for detecting a fungal infection in a woody plant comprising the steps of: use of X-ray tomographic images; obtaining a tomographic image of a stem of said woody plant comprising vasculature, said tomographic image comprising spatially distributed radiodensity measurements; characterized in that the method further comprises the steps of determining the presence or absence of regions in said image that have a radiodensity greater than a first predetermined radiodensity of the fluid generally found in the xylem of the plant vasculature; being the presence of said regions of higher radiodensity indicative of the presence of a fungal infection in the vasculature of the plant.

Description

DESCRIPTION

Method of diagnosis and treatment

Field of the invention

[0001] The invention relates to the diagnosis and treatment of fungal infections of woody plants, especially plants of the genus Vitis, Actinidia and Citrus and hybrids thereof, and especially fungal infections comprising tinder, dead black arm (fungi Botryosphaeriaceae), or infections similar to tinder, or Eutypa lata infections.

Background and prior art known to the applicant

[0002] Fungal diseases of wood are currently the most important threat to the lifespan of commercial fruit trees and grapevines worldwide. They jeopardize profitability and sustained production by reducing fruit yield and quality, primarily by inducing rot and death of infected plants. Key species affected by these diseases include grapevine (Vitis spp. And their hybrids), kiwi (Actinidia spp. And their hybrids), although it is increasingly recognized that there are similar diseases that affect many other hosts, as will be described. later.

[0003] In grapevine (Vitis spp. And its hybrids), tinder, Petri disease, Eutypa dieback and dead black arm (BDA) are the main wood diseases that induce said rot and the death of vine plants (Luque et al., 2009, "Symptoms and fungi associated with declining mature grapevine plants in northeast Spain", Journal of Plant Pathology 91,381-390). As effective control measures against these diseases are not available, they seriously affect the durability of the wine heritage (Larignon et al., 2009, "Esca et Black Dead Arm: deux acteurs majeurs des maladies du bois chez la vigne", Comptes Rendus Biologies 332, 765-783.).

[0004] Tinder is a complex disease of grapevine wood that is reaching a growing and unprecedented importance worldwide. Although it was originally thought to only affect mature vines, the disease is currently at high levels in many younger plants. It is accepted that tinder comprises a number of different diseases and that the main causative agents (mainly vascular fungi) invade the vines not only through damage in the field, but also as a result of practices in nurseries. When infection begins in the nursery, plants can develop a variety of diseases, ranging from Petri decline (also known as "black goo" or sticky black substance) to complete tinder, with or without white rot of woody tissues.

[0005] Tinder is produced by the presence of at least three fungi, Phaeomoniella chlamydospora, Phaeoacremonium aleophilum and Fomitiporia mediterranea, which act alone, in combination or successively. The first two fungi are responsible for vascular necroses, which appear as black spots on the wood, sometimes with some dark and hard areas of necrosis, while F. mediterranea (and / or other wood-rotting basidiomycetes) induce rot white or «amadou».

[0006] However, the pattern of symptoms and the initial appearance of their expression is highly variable: severely infected plants may remain asymptomatic in successive years for a series of years without visible symptoms (on the leaves) of tinder (tinder 'hidden »). This intermittent pattern of symptom expression results in infected plants being indistinguishable from healthy plants.

[0007] It is of utmost importance to ensure a healthy propagation of the vine in the nursery, where only material without staining on the wood should be selected. Once planted in the vineyard, the infected planting material develops decline or Petri disease, and is also characterized by dark spots on the wood, slow dieback, and “black goo.” Since these fungi grow inside the wood of the trunk and large woody branches, cannot be reached by spraying fungicides on the leaves, the best solution is still to dig up the infected vine and burn it.

[0008] The major problem with tinder comes from its initial stages: usually, the plants are externally asymptomatic, although occasionally they may present fruiting bodies and spores. However, they are capable of spreading the disease through common practices in vineyards, such as pruning. In other words, a conventional viticultural machine can pass the disease from an infected but asymptomatic vine to the following. The spores are also spread by the action of wind and rain. Therefore, although pruning tools and specialized viticultural machinery are not important in the spread of spores, they can play a role in the spread of fungal mycelia from diseased plants to healthy plants. However, when it comes to the spread and penetration of spores, pruning wounds and other injuries caused by specialized viticultural machinery are of particular importance. For all these reasons, it is of utmost importance to develop methodologies that can perform non-destructive screening and detect the initial stages of tinder, either in nurseries or in vineyards. Improved strategies and methods are also needed to treat such fungal infections at an early stage, while eradication of the disease is still possible.

[0009] Other plant species other than vine are also subject to diseases that affect wood, similar to tinder. In 1999, a wood rot of the kiwi climbing plant was observed for the first time (Actinidia) in Italian crops. A similar illness was subsequently reported in Greece and New Zealand. These diseases were described by the terms "similar to tinder" in France and "elephantiasis" in Italy.

[0010] Analysis of kiwi cultures showing visual symptoms of tinder-like disease revealed the presence of two types of necrosis in the wood: a) hard and brown rot beginning in the medullary tissues and gradually expanding throughout the trunk or branches, and b) soft white rot like "amadou wood" of the vine. Isolates from necrotic parts of wood showed various fungi, including: Mediterranean Fomitiporia, Phaeoacremonium aleophilum, Phaeoacremonium parasiticum, and Cadophora malorum. At least two of the exposed fungal species (F. mediterranea and P. aleophilum) are also implicated in vine tinder disease, and commonalities between kiwi rot and vine tinder can be assumed.

Citrus species (such as Washington navel orange, lemon and common mandarin grafted onto bitter orange rootstocks) are experiencing a serious decline caused by F. mediterranea in plantations in southern Greece. Affected trees show leaf chlorosis, defoliation, and twig and shoot death. Cross sections of the large trunks and branches reveal a rot in the center, which is surrounded by hard brown necrotic wood. Symptoms begin with pruning wounds and spread to the rootstock wood, being similar to the symptoms of tinder wood on the vine.

[0012] As in grapevine, kiwi and citrus, tinder-like diseases have also been identified in many other species. Phaeoacremonium aleophilum has been reported in Prunus species showing symptoms of regressive death in the Western Cape Province, South Africa. Serious symptoms of regressive death, caused by Pm, have been observed. parasiticum, on cherry trees in Greece. Phaeoacremonium mortoniae was shown to cause brown spots on the wood of Fraxinus pennsylvanica and Salix spp.

[0013] One of the main causative agents, Fomitiporia mediterranea, is also found in Corylus avellinus, Olea europaea, Lagerstroemia indica, Actinidia sinensis, Acer negundo, Olea europaea, Quercus sp., Quercus ilex, Ligustrum vulgare and Robinia pseudoacacia.

[0014] Another plant disease, and affecting woody species in the same way, is eutypiosis, also known as "Eutypa dieback", which is caused by the fungus Eutypa lata. E. lata is a vascular pathogen found in at least 88 species of woody dicotyledons (Carter, 1991. "The status of Eutypa lata as pathogen", Phytopathological Paper 32) and is responsible for significant economic damage, mainly in the vine and the apricot.

[0015] Many forest and ornamental species also host the pathogen, but it is not known with certainty if all isolates are pathogenic, although the pathogenicity of E. lata has been confirmed in vine, apricot, sweet cherry (Prunus avium L. .), Virginia cherry (Prunus virginiana L. var. demissa), cherry (Prunus cerasus L.), almond (Prunus dulcis), apple (Malus domestica Borkh.), pear (Pyrus communis L.) , the walnut (Juglans regia L.), the olive (Olea europaea L.), and Ceanothus L. spp.

[0016] In the vine, the most recognizable symptom of Eutypa dieback is the stunted appearance of the shoots in the early growth period, with small deformed chlorotic leaves and short internodes. However, the symptomatic expression varies markedly from year to year. Nondestructive techniques for the diagnosis of Eutypa regressive death are not yet available.

[0017] On the other hand, no effective treatment is available, and infected plants will die within a few years.

[0018] Another disease of interest, especially in grapevine (Vitis), is associated with infection by Botryosphaeriaceae fungi. These have a wide distribution and are commonly associated with dieback and cankers of woody and non-woody host plants, including forest trees, e.g. ex. Pinus spp., Eucalyptus spp., Quercus spp., Olea, drupe trees, Protea and vine.

In the vine, several species of Botryosphaeriaceae are associated with wood necrosis and are also capable of infecting both young and mature tissues, as well as green shoots, producing cankers, vascular discoloration and / or other types of dark spots in timber.

[0020] Some species of Botryosphaericaeae are responsible for dead black arm disease (BDA), the symptoms of which in wood include V-shaped necrosis, similar to those caused by Eutypa lata, and brown longitudinal spots throughout the tissues. affected. Other symptoms similar to those of Eutypa dieback are usually observed, such as stunted chlorotic buds or deformed leaves with necrotic areas.

[0021] It can be appreciated that all these diseases share common characteristics: (1) they are mainly woody plant diseases, and especially woody fruit plants of commercial importance; (2) are of fungal origin, and (3) pathogens invariably colonize the plant vasculature, especially the xylem. On the other hand, since (4) the symptoms of the disease are often localized (at least in the early stages) within the woody parts of the plant, it is difficult, if not impossible, to make a non-invasive diagnosis of the conditions. Furthermore, once detected, (5) the treatment of the disease is difficult, since the internalization of the fungal infection makes it less susceptible to external application of fungicides.

[0022] In the broader context of diseases of commercially important woody parts of plants, one of the biggest problems facing agriculture is the control of pathogens and insects that thrive inside the wood and therefore in a location that conventional fungicides / insecticides cannot reach. Therefore, it is extremely important to develop methods to detect the existence and location, within infected plants, of wood diseases during the initial but asymptomatic and contagious stages of infection (and therefore during an early stage of infection). potentially treatable) and to develop treatment regimens that are effective against such diseases. Addressing these problems is among the objectives of the present invention.

Summary of the invention

Accordingly, the inventors provide a method for detecting fungal infection in a woody plant according to claim 1 appended hereto. A wide range of tomographic imaging methodologies are available, including X-ray, PET (positron emission tomography), electrical resistance tomography, and acoustic tomography (including ultrasound). However, the inventors have discovered that the use of X-ray tomography techniques, including computed axial tomography (CT), microcomputed tomography, tangential scan, plain radiography, and simple multi-angle radiography, allows to diagnose externally asymptomatic plants with a wide range of fungal infections.

In this sense, the present method of the invention offers the possibility of detecting a fungal infection in said plant, at a stage in which it is possible to apply a treatment methodology at an early stage, that is, when the treatment is possible. If such diseases remain undetected and therefore untreated, the plant will normally die, and it will be a source of infection that can spread to other plants.

Although tomographic images have been used to image wood rot caused by fungal rot of dead woody material, and also late stages of rot in dead zones of woody material in tree trunks in growth, the present invention relates, instead, to the early detection of fungal infection of live growing plants susceptible to said infection by phytopathogenic fungi. For example, US patent document 4,283,629 in the name of Habermehl describes a tomographic technique to detect "red rot" in trees, in which dead internal woody areas on tree trunks can be detected by reducing the radiodensity in comparison with the surrounding healthy areas. The methods presented in this publication are geared towards the commercial production of construction lumber, to aid in making business decisions about when a lumber tree should be felled, and to what extent its woody parts can be used in various manufacturing processes. such as the production of cellulose or wood for construction.

[0026] In particularly preferred embodiments of the present invention, said woody plant is a plant of the genus Vitis or a hybrid thereof, a plant of the genus Actinidia or a hybrid thereof, or a plant of the genus Citrus or a hybrid of it.

[0027] In any aspect of the invention, it is also preferred that said fungal infection is a fungal infection that causes an accumulation or modification of fluid in the xylem of said plant. This fluid, like the "black goo" seen in tinder infection, results in a particular characteristic response on the tomographic image, allowing it to differentiate itself from other internal features, such as fungal rot.

[0028] Accordingly, it is especially preferable that said fungal infection comprises tinder or a tinder-like disease.

[0029] In other preferred aspects of the invention, said fungal infection comprises infection by Eutypa lata, or cankers associated with Botryosphaeria (or generally infection by fungi of the Botryosphaeriaceae family), or infection by Phoma tracheiphila.

In any aspect of the invention, it is especially preferable that said tomographic images comprise X-ray axial tomography. The inventors have shown that X-ray axial tomography is especially effective in the identification regions of plant material, especially those that They are not visible on the outside of the plant, which are colonized by phytopathogenic fungi.

According to the invention, the inventors provide said method using X-ray tomography, and especially X-ray axial tomography or X-ray microtomography, where said method comprises the steps of: obtaining a tomographic image of a stem of said woody plant, said tomographic image comprising spatially distributed radiodensity measurements; determining the presence or absence of regions in said image that have a radiodensity greater than a first predetermined radiodensity; the presence of said regions of high radiodensity being indicative of the presence of said fungal infection.

[0032] Exceptionally, the inventors have discovered that such X-ray tomographic images reveal regions of living plant material that are colonized by fungal pathogens and, especially in cases where the pathogenic fungi colonize the plant vasculature with subsequent production of fluid in the vasculature or a chemical modification of fluid in the xylem (eg, through the production of polyphenolic material or gums), this is evidenced by regions of higher radiodensity. Pathogenic fungi tend to induce the production of radiodense material in the vasculature (that is, more radiodense than the fluid usually found, eg, in the xylem of the plant), resulting in a positive indication of such infection . Instead, a prior use of X-ray tomography to image rot in dead wood (eg, in building materials or living trees of urban areas) detects the rotten area as regions of very low radiodensity, producing, in fact, little or no signals compared to the signals produced in the absence of wood. Therefore, it is often not possible to differentiate the presence of rot in dead wood (eg, by soft rot fungi) from holes in the wood itself.

[0033] In said method, it is especially preferable that said first predetermined radiodensity is the average radiodensity of a corresponding region in an uninfected control plant. In this sense, the method can easily be adapted for application in a number of woody species by calibrating the tomographic images against images taken from healthy plants.

[0034] Preferably, in any such method, and especially in the case of grapevine, said first predetermined radiodensity comprises 100, 120, 130, 140, 150, 160, 170, 180, 190 or 200 Hounsfield units.

Hounsfield units are well known in the field of X-ray tomography, and are a linear transformation of the radiological attenuation coefficient in the material, adjusted with respect to the corresponding attenuation in air and water, namely:

where:

R is the radiodensity in Hounsfield units

Px is the linear attenuation coefficient in the sample

Pw is the linear attenuation coefficient of water

Pa is the linear attenuation coefficient of air

In other preferred embodiments, the inventors provide a method that further comprises the step of: determining the presence or absence of regions in said image that have a radiodensity lower than a second predetermined radiodensity; being the presence of the two regions of high and low radiodensity indicative of the presence of an advanced stage of said fungal infection. Preferably, said second predetermined radiodensity comprises -200, -300, -400, -500, -600, -700 or -800 Hounsfield units.

In any aspect of the invention, it is preferable that said radiodense regions have a spatial dimension of less than 5 mm, 2 mm, 1 mm, 500 microns, 200 microns, 100 microns or even 50 microns. The inventors have discovered that the radiodense regions correspond to the accumulation of a "black goo", mainly of plant origin, and possibly comprising an excretion of polyphenolic compounds secreted by the plant as part of a host defense mechanism. Fluid accumulates primarily in the xylem vessels of the plant, and thus the radiodense regions reflect the narrow and elongated nature of the vasculature. Therefore, at least one of the spatial dimensions (width, height and length) of the radiodense regions is small, as indicated above. The inventors have discovered that these regions can be imaged by the techniques described herein, and are especially indicative of tinder-related or tinder infections.

[0038] Such a method can be applied especially to fungal infections such as tinder or tinder-like infections, where the accumulation of radiodense material in the vasculature of the plant is followed, during the development of the infection, by the appearance from regions of dead tissue rot in the plant structure, such as the so-called amadou in vine tinder. In this sense, the presence of both regions of relatively high and relatively low radiodensity can detect an especially advanced stage of such a fungal infection. This can help make decisions regarding the corrective actions to be taken, such as treating the infected plant, or a decision to start up and replace the plant.

Also in any aspect of the invention, it is preferable that said stem has a diameter of between 1 and 10 cm. The rationale for the described testing methodology is to be able to treat infected woody plants, and especially vines, and the use of the technique on relatively small stems helps to identify the disease at an early stage.

Also in any aspect of the invention, it is preferable that said woody plant comprises graft or rootstock material, and that said stem has a diameter of less than 1 cm. The inventors have discovered that the techniques described herein allow for rapid selection of rootstock and grafting material, typically having stem diameters less than 1 cm, when they leave the nurseries. This is especially true in the case of vines, where it is essential to prevent infected material from leaving the vineyard nurseries, assuming a possible source of infection to other healthy plants. The availability of such methods to diagnose these devastating diseases in species of commercial importance leads the inventors to be able to provide strategies and methods that were not available until now to manage said diseases in woody species.

Accordingly, the inventors provide a method of controlling fungal infection in woody plants according to claim 12 appended hereto.

[0042] In any aspect of the invention, a series of fungicides are contemplated, and these can be selected by the person skilled in the art based on their efficacy against the specific pathogenic fungus to be treated. Among the families of fungicides available to those skilled in the art are copper-based products; anilinopyrimidines; azoles and non-azoles (ergosterol biosynthesis inhibitors); carboxamides; phenylpyrroles; phosphonates; pyridinylethylbenzamides and strobilurins from QoI.

[0043] Specific examples of such fungicides include cyprodinil, fludioxonil, pyraclostrobin, boscalide and mixtures thereof (cyprodinil fludioxonil and pyraclostrobin boscalide), tebuconazole and phosphonates.

[0044] There are also other compounds that show fungicidal activity, but are not specifically registered as fungicides, such as: sodium bicarbonate (NaHCO3) and potassium bicarbonate (KHCO3), and hydrogen dioxide (hydrogen peroxide).

[0045] Fungicides exhibiting plant growth stimulating activity, such as blad, are especially preferred. Blad is a polypeptide that can be extracted from the cotyledons of Lupinus albus, or that can be produced using recombinant DNA techniques. Details on blad are provided in pending international patent application WO2007010459.

[0046] Fungicides with a host defense inducing activity, such as chitosan, are particularly preferred.

[0047] Also especially preferred are fungicides that exhibit host defense inducing activity, in addition to some direct effects on fungi, such as hyaluronic acid (an elicitor for inducing systemic resistance, see World J Microbiol Biotechnol (2008) 24 : 1153-1158) or acibenzolar-S-methyl.

[0048] Preferably, said fungicide is injected into said plant. By "inject" it is understood that said fungicide is introduced directly into the internal region of the plant where the tomographic images have detected the fungal infection. Like the literal injection of said fungicide, other means can be used, such as drilling a hole in the stem or trunk of the plant and dispensing the fungicide inside. A slow release fungicide plug is considered especially advantageous.

[0049] In especially preferred embodiments of the invention, the tomographic image is used to identify the region of the plant in which the infection is localized (via the regions of relatively high radiodensity), and the fungicide is introduced directly into these regions.

Brief description of the drawings

The invention will be described with reference to the accompanying drawings, in which:

Figure 1 shows photographs of cross-sections in tinder-infected vine stems;

Figure 2 is a light micrograph of a vine infected with tinder;

Figure 3 shows photographs of "black goo" in the vasculature of the vine infected with tinder;

Figure 4 shows a photograph and a corresponding X-ray tomography of a vine infected with tinder;

Figure 5 shows photographs and corresponding X-ray tomographies of vine stems at various stages of tinder infection;

Figure 6 is a series of radiodensity scans and graphs on healthy vine stems;

Figure 7 is a series of radiodensity scans and graphs on tinder infected vine stems; Y

Figures 8 to 12 are X-ray microtomographic images of tinder infected vine stems.

Description of preferred embodiments

[0051] Figure 1 represents, photographically, a typical progression (from A to D) of the infection by tinder in vine wood together with some characteristic structural features of the wood in its diseased state. Figure 1A shows a section through a vine in its early stages of infection, showing black dots (a) on an apparently healthy wooden background. Figures 1B and 1C show intermediate stages of tinder infection, showing black spots (a) and affected brown wood (b), as well as scars (c) and pith (d), which are normal characteristics of the wood. Figure 1D shows final stages of tinder infection with dead wood (e) and 'amadou' (f). It can be clearly seen that, in the earliest stages of the infection (Figures 1A and 1B), the infection is located inside the woody parts of the vine. There are no external symptoms towards the periphery of the wood and, therefore, there are no visible signs of infection apparent in the plant.

[0052] Optical microscopy of a section of a vine infected with tinder and asymptomatic reveals (in figure 2) the presence of "black goo" in the lumen of the tracheids and vessels of the plant xylem (black arrows). The presence of this comparatively radiodense material is that which can be detected using tomographic images and especially X-ray tomographic images.

[0053] Figure 3 shows the presence of "black goo" both inside and oozing from the xylem vessels in a cut section of tinder infected vine wood. In Figure 3 (a), a peripheral region of the stem (B1) shows healthy and uninfected vine wood, while the dark region (B2) shows “black goo” within the xylem. In Figure 3 (b), the "black goo" is seen oozing (B3) from the xylem vessels.

[0054] Figure 4A is a cross-sectional photograph of a tinder-infected vine stem in an intermediate stage of infection, taken after several CT scans of an intact tinder-infected vine trunk. Figure 4B is a tomographic image obtained by X-ray axial tomography in accordance with the present invention. The tomographic image (4B) shows radiodensity according to the scale presented on the right side of figure 5D. Dark colors indicate regions of low radiodensity, with lighter areas indicating increasing levels of radiodense material. Black represents in the image a radiodensity of approximately -135 Hounsfield units, and white represents approximately 215 Hounsfield units. Images were obtained using a Philips TOMOSCAN AV computerized axial tomography apparatus. The images were obtained with the following parameters:

Scanner: 120 kV, 90 mA, 2 s

Tilt: 0.0 °

[0055] Depending on the size of the trunk from which images were to be taken, the following apertures were used:

250mm / 1.2

270mm / 1.1

160mm / 1.9

[0056] Figure 4B graphically represents the ability of X-ray axial tomography to detect signs of infection by tinder in the core of the woody material, by virtue of the "black goo" of radiodense material in the vascular system of the plant. The radiodensity values of two points in the image (184 and 246 Hounsfield units) are provided, indicating regions that present vasculature filled with the characteristic "black goo" of tinder infection.

[0057] Figure 5 (AD) represents photographs of cross-sections (column on the left) of grapevine wood with various degrees of infection by tinder, together with X-ray computed axial tomography images (column on the right) obtained before to section the wood to take pictures. The tomographic images show radiodensity maps of the wood, according to the scale adjacent to Figure 5D, with dark regions representing low radiodensity and light regions with a corresponding high radiodensity. Black regions have a radiodensity of -135 Hounsfield units or less, with white regions having a radiodensity of 215 Hounsfield units or greater.

[0058] Figure 5A shows images of a healthy and uninfected stem; Figure 5B is of a stem during the early and asymptomatic stages of infection. The regions of high radiodensity are especially noticeable (white areas in the radiodensity map of Figure 5B) that correspond to the black areas in the corresponding photograph, and that comprise regions of vascular tissue that present “black goo” within the xylem, as well as the areas of brown affected wood that surround them.

The images in Figure 5C correspond to a stem in an intermediate stage of infection by tinder. Again, the areas of high radiodensity represented as light colored regions correspond to the dark radiodense regions that comprise regions filled with "black goo" of the woody stem shown in the photograph.

Lastly, the images in Figure 5D correspond to a stem in an advanced stage of tinder infection. Like the regions of high radiodensity (the light-colored spots on the scan) corresponding to full vasculature, the images also show (on the left side) regions of low radiodensity (the dark or black regions on the scan) that correspond to areas affected by white rot or "amadou" shown in the photograph.

The images demonstrate the ability of X-ray computed axial tomography to identify, non-invasively, early stages of tinder infection by detecting regions of high radiodensity, as well as to identify later stages of the infection. infection by detecting regions of low radiodensity, corresponding to «amadou».

[0062] Figure 6 (a) - (e) shows radiodensity patterns obtained by X-ray based computed axial tomography of a healthy and intact vine stem. The graphical representation of radiodensity corresponds to the section illustrated in the 2-D tomographic image represented with a light-colored line. The point in the 2-D image is represented by the vertical reference line in each graph, with the corresponding horizontal line determining the radiodensity measurement of the point expressed in Hounsfield units.

[0063] In each graph, the illustrated radiodensity range is plotted on a scale of -478 to 173 Hounsfield units, and the "point values" are as follows:

The very low radiodensity of the point value of Figure 6 (c) corresponds to a region of fluffy pith material in the center of the stem. Higher values (eg, 119 and 125) correspond to normal radiodensity values seen in highly hydrated regions of healthy wood tissue.

[0065] Figures 7 (a) - (l) show radiodensity patterns obtained by X-ray-based computed axial tomography in three sections of a tinder-infected vine stem. The graphical representation of radiodensity corresponds to the section illustrated in the 2-D tomographic image represented with a light-colored line. Again, the point in the 2-D image is represented by the vertical reference line in each graph, with the corresponding horizontal line determining the radiodensity measurement of the point expressed in Hounsfield units.

[0066] In each graph, the illustrated radiodensity range and "point values" are as follows:

Very high radiodensity levels (usually above 250 Hounsfield in this particular example) correspond to regions where "black goo" has accumulated in the plant vasculature. Very low levels (usually negative values in this example) they correspond to regions corresponding to an "amadou", or to a hole or pith in the stem.

[0068] Radiodensity analysis of a large number of grapevine wood samples in which characteristics have been identified by subsequent sectioning has revealed the usual ranges of radiodensity observed in this species for various botanical characteristics, or characteristics of the infection. by tinder. These values are indicated in table 1.

Table 1

[0069] By using X-ray tomographic images, followed by subsequent sectioning and identification of features, those skilled in the art can easily determine the typical range of densities for features in other woody species and for other fungal infections. In this regard, the techniques described herein can be applied in a wide range of fungal species and infections.

[0070] Figures 8 to 12 show images obtained using high sensitivity X-ray computed tomography of vine stems (Vitis) infected by tinder at an early stage of infection, before external symptoms appeared.

[0071] Figure 8 shows a cross section (A) and a detail (B) of a thin trunk of a two-year-old vine infected with early stages of tinder. The scale bar represents 5 mm. The bright areas in the image represent regions of high radiodensity, indicative of tinder infection. The largest bright spot in inset (B) is approximately 250 microns in diameter, representing the surprising ability of the technique to visualize even early stages of tinder infection.

[0072] Figure 9 shows a longitudinal cross-sectional X-ray tomographic image of a thin trunk of a two-year-old grapevine (Vitis) infected with early stages of tinder. The scale bar represents 5 mm. The bright areas in the image again represent regions of high radiodensity, indicative of tinder infection. High radiodensity regions as small as 50 microns can be seen in the images.

[0073] Figure 10 shows X-ray tomographic images of xylem vessels with varying degrees of filling with the characteristic "black goo" of tinder infection. Images are taken on a vine trunk during the initial and asymptomatic stages of tinder infection The scale bar represents 100 microns, again representing that very small features related to infection can be identified.

Figures 11 and 12 are three-dimensional axial tomographic reconstructions of a vine trunk (Vitis), highlighting its irregular shape. The scale bar represents 10 cm in figure 11 and 10.13 cm in figure 12.

Claims (14)

1. Method to detect a fungal infection in a woody plant comprising the stages of: use of X-ray tomographic images; obtaining a tomographic image of a stem of said woody plant comprising vasculature, said tomographic image comprising spatially distributed radiodensity measurements; characterized in that the method further comprises the steps of determining the presence or absence of regions in said image that have a radiodensity greater than a first predetermined radiodensity of the fluid generally found in the xylem of the plant vasculature; being the presence of said regions of higher radiodensity indicative of the presence of a fungal infection in the vasculature of the plant. 2. Method according to claim 1 wherein said woody plant is one of: • a plant of the genus Vitis or a hybrid thereof; • a plant of the genus Actinidia or a hybrid thereof; • a plant of the genus Citrus or a hybrid thereof. 3. Method according to any of claims 1 or 2 wherein said fungal infection causes accumulation of fluid in the xylem of said plant. 4. Method according to claim 2 wherein said fungal infection comprises one or more of • tinder or a tinder-like disease; • Eutypa lata infection; • cankers associated with Botryosphaeria; • Phoma tracheiphila infection. Method according to any preceding claim wherein said tomography is X-ray axial tomography or X-ray microtomography. Method according to any of the preceding claims, wherein said first predetermined radiodensity is the average radiodensity of a corresponding region in an uninfected control plant; or said first predetermined radiodensity is 100, 120, 130, 140, 150, 160, 170, 180, 190 or 200 Hounsfield units. Method according to any of the preceding claims, further comprising the step of: determining the presence or absence of regions in said image that have a radiodensity lower than a second predetermined radiodensity; being the presence of the two regions of high and low radiodensity indicative of the presence of an advanced stage of said fungal infection. 8. Method according to claim 7 wherein said second predetermined radiodensity is -200, -300, -400, -500, -600, -700 or -800 Hounsfield units. Method according to any one of claims 6 to 8, wherein said radiodense regions have a spatial dimension of less than 5 mm, 2 mm, 1 mm, 500 microns, 200 microns, 100 microns or 50 microns. 10. Method according to any of claims 6 to 9 wherein said woody plant comprises a stem with a diameter of between 1 and 10 cm. Method according to any one of claims 6 to 9 wherein said woody plant comprises rootstock or graft material, and comprises a stem with a diameter of less than 1 cm. 12. Method of control of fungal infection in woody plants that includes the stages of: determining the presence or absence of said fungal infection using a method according to any one of claims 1 to 11; Y treating said woody plant with a fungicide, and preferably a systemic fungicide, if said fungal infection is thus detected. 13. Method according to claim 12 wherein said fungicide is injected into said plant. Method according to any of claims 12 or 13 wherein said tomographic image is used to identify the location in the plant where said fungal infection is present, and said fungicide is introduced at that location.