Evidence is accumulating that root system collapse is involved with HLB-induced tree decline, especially with commercial sweet orange and grapefruit trees on Swingle and Carrizo. Maintaining root health is imperative for keeping trees productive in an HLB endemic environment. In an effort to improve tree health by focusing on the roots, we have been experimenting with polymer coated nutrients and more recently TigerSul micronutrients in the field and greenhouse. In the greenhouse, Orange 15 rootstock were side grafted with HLB-infected Valencia sweet orange. Treatments were established with 10 replications each. Control treatments received either 1) bi-annual Harrell’s UF mix, or 2) bi-weekly liquid fertilizer. Experimental treatments received bi-annual treatments of the following: Harrell’s UF mix supplemented with bi-annual treatments of 3x overdoses of individual polycoated essential minor elements , TigerSul micro-nutrients , or 2x overdoses of the individual polycoated essential macro-nutrients. The experiment was carried out for one year. Effects on tree health, tree growth, root mass, SPAD, leaf and root nutritional analysis, and leaf and root Liberibacter titers will be presented. There were clear differences among treatments. Several treatments significantly improved tree growth and health as compared with the controls,drainage collection pot especially the 3x TigerSul manganese and 3x polycoated sodium borate treatments.
Results suggest that trees in the HLB world have higher specific micronutrient requirements than what are currently being recommended. In the field, we have several experiments that involve controlled release, ground applied fertilizers; these include the St. Helena project near Dundee, where sweet orange trees on numerous rootstocks are being grown completely with CRF and daily irrigation. The trees will be 7 years old in April, and they are now more than 80% infected with HLB, yet the large majority of the trees across all rootstocks have remained productive. Evolution of the CRF formulas and effects on tree health from St. Helena and other minor field CRF experiments will be discussed. It is clear that a constant supply of nutrients year-round is required to maintain productivity in the HLB world. Additional fine tuning of fertilizer composition, type, and delivery method should lead to improved tree health and productivity. Improved ground nutrition will continue to play a key role in integrated approaches to controlling HLB. Genetic variability for HLB tolerance/resistance is being identified in existing experimental rootstock germplasm planted throughout Florida, with both sweet orange and grapefruit scions. New rootstocks are being identified in these trials that show a reduced infection frequency, and less severe symptoms once infected, as compared to commercial rootstocks. Rootstocks showing promise include complex tetraploids, diploid citranges, and diploid pummelo x mandarin hybrids. Several of these rootstocks have been ‘Fast Track’ released for large-scale commercial evaluation. Current focus is on the identification of rootstocks that can sustain or increase productivity under heavy HLB pressure.
Data on these promising rootstocks will be presented. The fact that there is genetic variability in rootstock germplasm not pre-screened for HLB tolerance/resistance suggests that even greater progress can be made by focused selection, especially from crosses utilizing emerging HLB tolerant/resistant parents. Thus, we have adjusted our rootstock breeding/greenhouse screening program to focus on HLB by developing the ‘Gauntlet’ screening program described below. Following a preliminary calcareous soil/Phytophthora screen, selected individual hybrid rootstock candidates are transferred to citripots in commercial potting soil. Tops of these trees are propagated by rooted cuttings to produce seed trees on their own roots. The remaining individual liners are grafted with HLB-infected budsticks of Valencia sweet orange. The remaining rootstock top is then removed, forcing flush from the HLB-infected budstick. Trees are monitored for HLB symptoms, and healthy appearing trees are entered into a ‘hot psyllid’ house until psyllid feeding damage is observed on their leaves , followed by field planting at a challenging field site .The oldest ‘Gauntlet’ trees have now been in the field for approximately 2 years, and 20 promising new rootstocks have been identified so far. Our goal is to develop rootstocks that will facilitate sustainable and profitable citriculture in an HLB-endemic Florida, and possibly eliminate the need for psyllid control. Huanglongbing is one of the most devastating citrus diseases in the world. In Florida, it is associated with a bacterium Candidatus Liberibacter asiaticus and transmitted by a psyllid, Diaphorina citri Kuwayama.
We tested D. citri collected in many different venues over a period of 6 years for Las by molecular methods. Results surprised us. first, positive D. citri can be found long before symptoms develop on the plants at the site. Second, positive psyllids can ride on unprocessed fruit in trailers, even when there is no foliage. Third, about 10% of psyllid samples collected from plants for sale in Florida tested positive for Las. Finally, our data, and a related mathematical model, predict a form of transmission of Las that vastly increases the potential for spread of HLB. The mechanism now is known. The increase of infected vectors follows the growth of the insect population, independent of the incubation period in the plant. The implications of this new mechanism completely change our understanding of the epidemiology of HLB. It is possible to have positive D. citri throughout a grove before ever seeing a symptomatic plant. This mechanism has profound implications for disease spread, epidemiological research, early detection, long range dispersal, and grove management. Through utilizing the nutrient-rich phloem sap, sap feeding insects such as psyllids, leaf hoppers, and aphids can transmit many phloem-restricted pathogens. On the other hand, multiplication of phloem-limited, uncultivated bacteria such as Candidatus Liberibacter asiaticus inside the phloem of citrus indicates that the sap contains all the essential nutrients needed for the pathogen growth. Genome sequencing studies revealed that CLas can metabolize many sugars and amino acids found in the phloem sap. In addition, CLas can act as energy parasites and scavenge ATP from its host through the use of an ATP/ADP translocase. Furthermore, reduction in some minerals such as Zn and P in CLas-infected trees indicated that these minerals are required for the growth of CLas. The presence of gene in CLas genome also indicated that CLas can import Zn from its host. The phloem sap composition of many plants has been studied; however,drainage pot available data about citrus phloem sap is limited. In this study, we investigated the phloem sap composition of sweet orange. The phloem sap collected by EDTA or centrifugation method was derivatized with three different reagents and analyzed with GC-MS revealing 20 amino acids, 8 sugars, and 8 organic acids. Analysis of citrus phloem sap by inductive coupled plasma showed that it was rich in potassium, calcium, phosphorus, magnesium, and sulfur. Trace amounts of iron, copper, zinc, and boron were also detected. The ATP concentration in citrus phloem was 24.0 ± 4.0 ppm. Analysis of citrus phloem sap high performance liquid chromatography showed that citrus phloem sap was rich in nucleotides. Studies on seed transmission of ‘Candidatus Liberibacter asiaticus’ , the bacterium associated with Huanglongbing , described seedlings from infected trees which showed an abnormal growth phenotype but were free of CLas. We germinated populations of seeds of ‘Hamlin’, ‘Ridge Pineapple’, and ‘Valencia’ sweet orange from infected trees and observed chlorotic and stunted seedlings which failed to grow substantially. Tests detected no CLas DNA in these seedlings. These aberrant phenotypes were similar to symptoms expressed by infected trees, and persisted for 6 months or longer, at which point seedlings died or began to grow normally. Normal growth also was induced by grafting stunted seedling apices to healthy citrus seedlings. The absence of infection and their HLB-symptomatic phenotype suggest that extracellular factors produced by CLas in the mother tree affected the normal development of these seedlings, possibly through the alteration of normal gene expression. The similarity of these seedlings and symptomatic foliage suggest these factors are involved in disease development in infected trees. We propose these seedlings are a model system for understanding the molecular basis of symptom development in HLB-affected citrus trees.As Huanglongbing has continued to spread across the state, there is a growing body of evidence to support that in some instances, there are some off-flavors that can be detected in juice coming from fruit of HLB-infected trees compared to juice coming from the fruit of healthy trees.
The differences tend to be subtle but detectable in sensory evaluations by trained panelists, and in some instances by untrained panelists. Among the descriptors that have been used to describe HLB juice are bitter, sour, astringent, metallic, salty, umami, less orange aroma, less orange mouth feel, etc. Similarly, multiple studies have also identified several juice quality parameters, metabolites, or compounds that change with HLB infection; but, in all cases it has been changes in levels and not the presence or absence of a specific chemical. For instance, juice from HLB-infected fruit results in lower Brix, higher acid, and higher levels of limonin and nomilin. However, there is no one “smoking gun” parameter that can be measured that would indicate the potential for off flavors. In the present study, 14 different parameters were evaluated that encompassed sensory descriptors , basic juice chemistry , secondary metabolites , and harvest associated parameters . Samples were collected at random from fruit loads delivered to a commercial processing plant and were extracted using commercial equipment by the “USDA/State” lab that exists at commercial processing facilities. Sensory data were collected through the use of an electronic tongue. Basic juice chemistry data were collected using traditional wet chemistry techniques. Secondary metabolites were measured by HPLC and harvest date and fruit size were collected at the processing facility. Samples were collected from both Hamlin and Valencia varieties. Principal component analysis was used to reduce the number of variables considered for each of the varieties and the results were compared to a novel qPCR method to cross check the conclusions. Initial results indicated that a subset of parameters that include some sensory, basic juice chemistry, secondary metabolites, and harvest data can be used to identify fruit loads that have the potential to produce off flavors in Hamlin and to a lesser extent in Valencia varieties.Since its discovery in Florida in late 2005, citrus Huanglongbing , widely recognized as the most serious disease of citrus, has caused havoc in the Florida industry. Production costs have increased and production is down. Despite the apparent advances that growers and researchers have made in mitigating some of the symptoms of the disease, the statewide production continues to go down. All of the variables that go into the state forecast, tree number, fruit size, fruit per tree, and percent fruit drop are all trending in the wrong direction. This has led some growers to hold back on replanting in recent years which has further exacerbated the problem. The big question is how much lower will production go? Based on what we know, or in some cases based on our best guess, a model was created to estimate where we are in the HLB decline curve in the Florida industry. The model assumes that the industry focus will continue on the track of symptom mitigation instead of inoculum removal and management. Based on some conservative assumptions used in the model, the significant losses that have occurred the last several years should have been predicted. Going forward with the same assumptions, it appears that the production decline is near its predicted lowest significant drop and future losses will track the rate of replanting. That is to say, if tree attrition is greater than the rate of replanting, production will continue to decline. If the replanting rate is equal to or greater than the attrition rate, then the production will hold or increase slightly over time. Citrus is one of the most economically important and extensively grown fruit tree crops worldwide. Citrus production in most citrus producing countries, e.g., US, Brazil, and China, is facing an unprecedented challenge caused by Huanglongbing . Currently, no effective HLB management is available. Development of HLB resistant or tolerant citrus will provide a long-term, effective, and sustainable solution to HLB. Traditional plant breeding is unlikely to lead to HLB resistant or tolerant plants due to the lack of resistant varieties. Targeted genome engineering is expected to contribute significantly to future varietal improvement. Genome editing technologies using zinc finger nucleases , transcription activator-like effector nucleases , and clustered regularly interspaced short palindromic repeat /Cas9/single guide RNA have already been successfully used to genetically modify plants. Here, we reported our progress in modifying citrus genome suing Cas9/sgRNA technology.