Leaf Structure And Attenuation Coefficients Of Citrofortunella microcarpa Leaves Using A Portable TD-OCT System

Abstract

Citrofortunella microcarpa (C. microcarpa) is a native citrus widely spread in Southeast Asia but it is popular only in the Philippines [1].  Different common names are given for this species such as calamondin, kalamondin, kalamunding, kalamansi, calamansi, limmonsito and agridulce [2] and, in the Philippines, it is known as calamansi. It is used in culinary purposes [2–4], medical applications [5], and is an important commercial fruit crop in the country ranking fourth to banana, mango, and pineapple [6].  In the citrus industry, it is one of the most important sources of income to farmers but, since 2008, there is a decrease in area and production. Various diseases can infect calamansi and among them Huanglongbing, locally known as Leaf Mottling, the most severe and devastating disease [6]. The diseased leaves have normal size but have discoloration or blotchy mottle [7] which leads to a decrease in production. Traditional method used by farmers in diagnosing plants is through visual examinations and usually this is possible only when the plants suffered major damage already and cannot be save.  Early diagnosis is essential for the control of the disease. Polymerase Chain Reaction (PCR) and histological analysis are the commonly used techniques in agriculture for identifying infections and disease in plants [8,9].  PCR analysis is costly, destructive, and time-consuming.  There is also a DNA-based test [10] which is not reliable in asymptomatic stage, and the Iodine-Based Starch test [11] that is rapid and simple but will require cutting the part of the leaf to be immersed on the iodine solution. Other techniques are utilized that are not destructive and at the same time creates tomographic images of the sample such as the magnetic resonance imaging (MRI), X-Ray, confocal microscopy, fluorescence spectroscopy, scanning electron microscopy, and transmission electron microscopy [12–16]. The drawback with these techniques is the high cost of the equipment.  An inexpensive and non-invasive technique of diagnosing diseases in plants like the C. microcarpa is very much needed in an agricultural country such as the Philippines.

Optical Coherence Tomography (OCT) is a non-invasive, non-destructive optical imaging technique that uses a low coherence interferometer to obtain real-time cross-sectional images and three-dimensional images of a biological tissue sample. It was first developed in 1991 by David Huang [17] and OCT is notably used in biomedical applications including ophthalmology and dermatology [18–26]. It also extends to industrial, pharmaceutical, and poultry applications [27–31].  In the field of agriculture, OCT has in diagnosis of various infections or diseases in onions, melon seeds, kaki plant leaves, apple leaves, and orchids were analysed to control and limit the spread of diseases [32–41].  To our knowledge, this technique has never been applied in the Philippines.

Using a time-domain optical coherence tomography (TD-OCT) system the leaf structure and the upper epidermal thickness of Citrofortunella microcarpa leaves were examined and the attenuation coefficients were obtained.  These properties were used for differentiating healthy and unhealthy leaves. 

Shown in fig.1 is the schematic diagram of the portable TD-OCT system and the specification is summarized in table 1.  The TD-OCT system’s major components are the light source, the interferometer, and the receiver circuit.  The broadband light source is a Super Luminescent Diode (SLD) with a wavelength, l_O, of 1310 nm, and a spectral bandwidth, Dl, of 106 nm. A detailed description can be found in  [31, 41–43] The interferometer system consists of a 2x2 fibre coupler assembly with two collimators, and the optical path scanning mechanism.  The light coming from the SLD is split evenly into the sample arm and into the reference arm by the fibre coupler.  The two collimators of the fibre assembly are for the reference beam travelling to the scanning mechanism and the sample beam directed on the surface of the target.  The scanning mechanism is described in detailed in [42].  It consists of corner reflectors, a rotating disk, and an outer mirror.  It produces a scanning depth of 12 mm when the corner reflector rotates at 10 mm scanning radius and 25scan/s.

Ten leaves were taken from a C. microcarpa plant grown on a pot.  The OCT sample probe was placed on a fixed holder and was oriented vertically so that light is directed downward onto the sample leaf to obtain the depth profile (A-scan) which provides information on the leaf structure.  Assuming homogeneity within the layers, the attenuation coefficient was obtained by linear fitting the logarithm of the A-scan.

Figure 2 shows the box plot of the obtained attenuation coefficients for both the healthy and unhealthy leaves and the two different layers of the leaves.  The mean is shown as the horizontal line within the box.  For both layers of the leaves, the obtained mean μ of the healthy leaves were higher compared to the unhealthy leaves.  The attenuation coefficients of the epidermal and palisade layers of the healthy leaves were 27.07/mm and 15.36/mm, respectively.  The unhealthy leaves have a lower attenuation coefficient of 21.82/mm and 8.01/mm for the epidermal and palisade layers, respectively.  A comparison of the A-scan profiles obtained for the healthy and unhealthy leaves showed that there is a mixing of the epidermal and palisade layers within the unhealthy leaves.    These observations and the decreased in the attenuation coefficients of the unhealthy leaves are indications of deterioration on the photosynthetic rate that enhances the growth and development of the plant.  Our portable TD-OCT system can provide a non-invasive method and simple point measurement on the surface of the C. microcarpa leaves that can provide early detection that the plant is unhealthy.  The utilization of our system will benefit Filipino farmers.

Fig.1. Schematic diagram of the experimental set-up.

Fig.2. Box plot of the attenuation coefficients of healthy and unhealthy C. microcarpa leaves. Median is shown as the horizontal line within the box.

Table 1. Specification of Portable Fibre-based TD-OCT System