In 2014–2015, among fruit, oranges were in third place in the gross income of Puerto Rico, contributing $
6,452,000 to the island’s economy [1
]. However, in the last few years, the production of citrus on the island has decreased remarkably. Citrus greening (CG) disease, caused by the bacteria Candidatus Liberibacter asiaticus
), has become the principal cause of the worldwide reduction of citrus production [2
]. Citrus trees infected with CG present acidic fruits, asymmetric foliar chlorosis, diminished life cycle and, eventually, the tree presents dieback [2
]. In Puerto Rico, the citrus psyllid Diaphorina citri
(vector of CG) was first detected in 2001, while the presence of the bacteria, thus the presence of CG, was first detected in 2009 [4
]. Since then, there has been a reduction in citrus production, from 3500 tons in 2014 to 1080 tons in 2016 [6
]. Biological control, harsh pesticide treatments and thermotherapy are some of the controls that are being implemented to control either the vector or disease of CG [7
]. Another recommendation to maintain a stable citrus production is the elimination of advanced diseased trees, applications of intensive fertilizer practices, production of citrus trees inside protected structures and on-site planting of disease-free citrus trees [10
Grafted trees produced inside protected structures have the advantage of producing strong, disease-free trees, which can help fight the loss of citrus trees in the field caused by CG disease [11
]. The production of healthy citrus liners in containers inside of a protected nursery facilitates the development of a vigorous root system that results in a stronger/healthier plant [12
]. Substrates, fertilizers and hormones can help accelerate the growth of citrus rootstocks, and the combination of rootstock/scion can help shorten the time until transplanting to the field and provide some measure of resistance to diseases [13
Rootstocks not only provide a supportive root system, they also provide a better absorption of nutrients and water. They can also contribute characteristics to the resulting tree, such as tolerance to low temperature, high salinity, flooding and varying soil pH, and resistance/tolerance to nematodes and diseases [14
]. Of the multiple citrus rootstocks used in the industry, we evaluated Carrizo citrange and Swingle citrumelo. Carrizo citrange (Citrus sinensis
x Poncirus trifoliata
) has shown resistance to damage by Phytophthora
and citrus tristeza virus (CTV), an elevated resistance to the Tylenchulus semipenetrans
nematode and some resistance to the CG disease [15
]. Swingle citrumelo (Citrus paradise
Macf. x Poncirus trifoliata
) has presented good adaptation to sandy soils; a high germination percentage; an extensive radicular system; and moderate tolerance to salinity, boron toxicity, and CTV [16
]. Rootstocks help solve problems with soil, climate, pests and diseases, and can also influence the quality of production of the chosen scion [18
]. It is important to choose a good rootstock/scion combination [18
]. For the scion, we evaluated the variety Rhode Red Valencia. This orange presents intense color, low acidity, low vitamin C and 400% more antioxidants in comparison to other oranges [19
Not only the combination of rootstock/scion can reduce the time of development of the tree—the substrate can also affect the time of development [20
]. With the ideal substrate mix for a crop, one can therefore provide the optimal environment for the growth of an extensive root system [21
]. The citrus plant root system is limited, which results in a poor capacity for nutrient absorption [23
]. Therefore, a way of aiding the necessities of the citrus plant is through the components of the substrate mix and the fertilizer [23
Many substrate components are used at the nursery level and, for this research, a few of them were chosen for evaluation: peat moss, river sand, coco peat, coffee compost and rice husk. Peat moss is the result of decomposition of vegetable matter in an anaerobic environment; it usually exhibits a pH of 5.3–6.5 and more than 80% organic matter [24
]. River sand, or any sand, is frequently used to provide weight to the pot. Usually, it exhibits a neutral pH and a low capacity for cationic exchange [22
]. Coco peat is shredded coconut husk. Some characteristics of this substrate are: a pH that ranges from 5.3–6.3, high electric conductivity and water retention of 700–1100% [24
]. Compost, in general, is the transformation of organic matter that results in a new material that can favor oxygenation of the soil and substrate and promote higher root growth [28
]. Rice husk is the product of processing rice, and as a substrate it can provide higher air porosity [29
]. In Puerto Rico, little to no research has been done with these materials.
2. Materials and Methods
The experiment was conducted inside a nursery at the Agricultural Experiment Station (AES) in Corozal, Puerto Rico. The nursery was protected with a mesh (0.24 mm × 0.75 mm, Empresas San Pablo, Lares, PR, USA) to avoid entrance of the psyllid and large insects. The biosecurity protocol to enter the nursery consisted of using a lab coat and hair net and disinfection of footwear with sanitizing solution (sodium hypochlorite (NaClO) at 10%) to avoid the entrance of pathogens and insects. The first door of the nursery had an air curtain (48”, Mars Airs Doors, Gardena, CA, USA), which automatically started when the door was open. When the first door closed, the second door could be opened to enter the nursery.
Two citrus rootstocks were evaluated: Carrizo citrange and Swingle citrumelo. The seeds were obtained from the rootstock collection at AES, Corozal. The seed were germinated in a commercial mix (Pro-Mix BX with mycorrhizae) on 72-hole germination trays. After 3 months, on 14 April 2015, the seedlings were transplanted to 10.2 × 30.5 cm (4 × 12 inch) square pots (CP412CH, Stuewe & Sons. Inc., 31933 Rolland Drive, Tanget, OR, USA) containing the different substrate and fertilizer treatments. All the substrates were sterilized with water vapor (100 °C) for 4 h at AES in Adjuntas. The commercial mix used was Pro-Mix Sunshine Mix #4. This product contained Canadian peat moss, dolomitic lime, coarse grade perlite, gypsum and Sun Gro’s long-lasting wetting agent. The coco peat used was a commercial block made of coconut husk, compacted and imported by Empresas San Pablo, Lares, PR. The coffee compost was obtained from the AES in Adjuntas and had 4.89% nitrogen, 0.46% phosphorus, 3.33 mS/cm electric conductivity (EC) and a pH ranging from 6.72 to 6.84 [31
]. The rice husk was obtained from a rice planting in the Department of Agriculture. The sand used was river sand obtained from Corozal, Puerto Rico. The fertilizer quantity per treatment was calculated from the low (F1—5.6 g) and high (F2—8.5 g) recommendations of the product Caliber Cote 18-6-12 (Homogeneous Trace Pack, Helena Chemical Company, Collierville, TN, USA). There were two applications of fertilizers: at the transplant date or start of the experiment and 3 months later. The experiment was conducted from April 2015 to February 2016. The combinations of control media, substrates and the fertilizer quantities are described in Table 1
2.1. Evaluation of Rootstock
2.1.1. Height and Diameter
The height (cm) of each plant was taken from the top of the substrate to the petiole of the newest leaf every 2 weeks for 6 months. Tree diameter was taken at 5 cm from the substrate with a caliper (digital caliper, ABSolute AOS Digimatic, Mitutoyo Corp., Takatsu-ku, Japan) every 2 weeks for 6 months.
Chlorophyll of the rootstock leaves was measured with a chlorophyll meter (Field Scout, Chlorophyll Meter with Data Logger, Spectrum Technologies) every 2 weeks. The leaves sampled were the newest, fully-opened leaves. On each plant, three measurements of the same leaf were taken, and an average was calculated.
2.2. Evaluation of Rootstocks Grafted with “Rhode Red Valencia”
After 6 months, the rootstocks were grafted with “Rhode Red Valencia” (Citrus sinensis L. Osbeck) Vegetative material of “Rhode Red Valencia” was obtained from trees kept inside protected structures at AES at Isabela, Puerto Rico. The height of the developing scion was measured from the grafting site to the petiole of the newest leaf.
The same instruments and procedure as explained before were used with the scion for chlorophyll.
2.2.3. pH and EC
Samples of all the substrate treatments were sent to the Central Analytical Laboratory of the University of Puerto Rico at the beginning of the experiment for chemical properties testing. The pH and electric conductivity (EC) were measured by the pour-through method [32
] (pH/EC/TDS Meter, Milwaukee, MW 802) every 2 weeks, from planting until the end of the experiment.
2.2.4. Dry Weight of Shoots and Roots of Trees Grafted with “Rhode Red Valencia”
At 3 months after grafting, the trees were harvested for dry weight. The division of the root and shoot was made at the top of the substrate. The dry weight of shoot and root were taken separately. The samples were dried in an oven (Oven PKN810, Yamato) at 70 °C for 144 h (6 days).
2.3. Experimental Design and Statistical Analysis
The design of the experiment was a randomized complete block with four blocks of six replicate trees of each treatment. The data was statistically analyzed using ANOVA and Fisher’s least significant difference (LSD) with a P < 0.05.