Global citrus farming is threatened by various phytopathologies that reduce productivity and, consequently, lead to higher prices of oranges and their derivatives. Therefore, for the development of our project, we focused on understanding more about the main diseases that affect two different stages of the orange production chain: pre-harvest and post-harvest.

After engaging with our stakeholders, we understood that Greening, Sour Rot, and Green Mold are the main diseases that need to be addressed in the current context. Based on this information, we conducted our theoretical research on the epidemiological aspects of these phytopathologies, such as incidence levels, biological cycles, transmission methods, and the specific plant tissues affected by the pathogens.

Greening

Greening or Huanglongbing (HLB) is a disease caused by Gram-negative bacteria of the genus Candidatus Liberibacter (CL). Three species of this genus are recognized as responsible for the disease: Candidatus Liberibacter asiaticus (CLas), Candidatus Liberibacter americanus (CLam), and Candidatus Liberibacter africanus (CLaf)^[1]. In Brazil, C. Liberibacter asiaticus is the predominant species, present in more than 99% of the infected citrus plants^[2].

The bacteria are transmitted by the psyllid Diaphorina citri, a grayish-white insect with dark spots on its wings, measuring between 2-3 mm in length, frequently found in orchards during the plant’s sprouting seasons. This vector is responsible for spreading the Asian and American species of Candidatus Liberibacter. Meanwhile, the African species of the bacterium is transmitted by another psyllid, Trioza erytreae^[1].

Disease History

The identification of the first case of Greening worldwide is difficult to determine. Historical records indicate that the disease was responsible for the decline of citrus crops in India during the 18th century. Initially, researchers believed that the Citrus Tristeza Virus (CTV) was the main agent responsible for this decline, but after more in-depth studies, it was found that the true culprit was a bacterium of the genus Candidatus Liberibacter^[1,3].

Since then, Greening has spread to multiple regions around the world (Figure 1). In Saudi Arabia, by 1986, all orange and tangerine trees were affected, with only limes remaining. In Indonesia, Greening destroyed 3 million trees, while in Africa, losses ranging from 30 to 100% of harvests were reported. In the Philippines and India, the disease continues to raise major concerns, emerging even after the replanting of new seedlings^[4].

In the Americas, the first record of Greening occurred in 2004 in São Paulo state, Brazil. However, a survey conducted just six months after the official detection, revealed infected trees in 46 municipalities in São Paulo, suggesting that the disease had been present in the region for approximately ten years. By August 2005, symptoms of Greening were also recognized in Florida, and currently, the eradication of Candidatus Liberibacter asiaticus is no longer considered possible in the U.S. state^[1,4].

Disease Symptoms

The symptoms of Greening can appear in different parts of the plant and, in later stages, become more difficult to identify. The period between infection and symptom appearance is not exactly known, but once visible, they can be observed in multiple plant organs^[1]. These manifestations may be present throughout the year, occurring most frequently between late summer and early spring^[2].

Initially, it is possible to observe the emergence of shoots with yellowish leaves, which over time become blotchy. Blotching is characterized by irregular spots on the leaf blade, alternating between green and yellow, with no symmetry between the two halves. Affected leaves tend to fall, while new small shoots emerge with vertically oriented leaves, forming a "rabbit ear"-like structure^[2]. In some cases, the leaf midrib becomes thickened, pale, and even rough to the touch. As the disease progresses, trees exhibit canopy thinning, dead branches, and discolored leaves, which contrast with the healthy regions of the plant^[1,2,4].

Fruits of infected trees do not ripen properly and show a blotchy light green color, falling prematurely. In addition, the fruits are smaller, deformed, and asymmetrical, with orange coloration near the peduncle insertion. When the fruit is cut open, orange streaks in the columella, aborted seeds, and a thicker-than-normal albedo can be observed. The juice from affected fruits has a more acidic, less sweet, and more bitter taste. The root system of infected plants is also compromised, with a reduced number of fibrous roots, suggesting a condition of nutritional deficiency (Figure 2)^[1,2,4].

In practice, the characteristic visual symptoms of Greening can be mistaken for those of other citrus diseases or specific nutritional deficiencies. For this reason, studies highlight the urgent need for effective and accurate methods to diagnose the disease^[16]. Many solutions found in the literature are based on genetic analysis; however, sample preparation and the high cost of reagents limit their practical feasibility^[16]. In response to these challenges, our team Pepcitrus has developed a hardware device designed to efficiently and innovatively diagnose plants infected with Greening.

Biological Cycle

The Greening bacterium is primarily spread and transmitted through infected plants and psyllids carrying the pathogen (Figure 3). As it lives in the plant’s “veins” (phloem vessels), the bacterium can quickly disseminate throughout the entire tree, including roots, branches, leaves, and fruits. By the time symptoms appear at branch tips, the pathogen has already spread through the entire plant, including the lower trunk and root system. Consequently, pruning symptomatic branches is ineffective and potentially harmful, as it does not cure the plant, and the new shoots that emerge after pruning attract psyllids, which transmit the bacterium to healthy plants, leading to new infections^[2].

The psyllid, which undergoes six developmental stages, acquires the bacterium by feeding on infected plants and transmits it when feeding on healthy ones. Acquisition and transmission of the bacterium occur after approximately 15 minutes of feeding, and the longer the psyllid feeds, the higher the transmission rates. The nymph stage is the most efficient one for acquiring the bacterium, while transmission is more intense in shoots than in mature leaves. Therefore, the current measure is to remove diseased trees as soon as they are identified, even though this may result in production losses for the orchard. This is because the trees can act as reservoirs for the disease, and psyllids feeding on them can spread the bacteria even further.. To protect healthy plants, it is necessary to apply insecticides frequently, as psyllids prefer to feed and reproduce on shoots^[2].

In addition, the bacterium can also be transmitted through grafting with infected buds and by contaminated nursery plants. Unlike other diseases, the Greening bacterium is not spread by seeds, wind, water, or agricultural tools. After the bacterium is transmitted, Greening symptoms begin to appear on the leaves approximately four months after infection and continue to manifest throughout the year. Even asymptomatic infected plants can serve as a source for the pathogen dissemination. Therefore, it is essential that orange trees are regularly inspected by trained professionals to ensure that, until new technologies capable of directly combating the bacteria become available, diseased plants are promptly removed and the spread of the disease is effectively controlled.^[2].

Green Mold

Green Mold is the main post-harvest disease affecting citrus production worldwide. Post-harvest diseases are estimated to cause around 50% of total citrus losses, and Green Mold alone accounts for approximately 90% of these post-harvest losses^[10,12]. The pathogen responsible for the disease is the fungus Penicillium digitatum, which infects injured oranges and initiates the degradation of the plant cell wall, leading to fruit softening and decay (Figure 4)^[11].

Disease Symptoms

Infected fruits display characteristic and easily recognizable symptoms, including initial softening and water-soaked areas on the peel, followed by the development of a white mycelial growth that later produces abundant green conidial masses.^[12].

Biological Cycle

The disease cycle begins when Penicillium digitatum spores enter citrus fruits through wounds caused by harvesting, handling, or insect damage. Once in contact with the injured tissue, the conidia germinate rapidly, forming germ tubes that penetrate the pericarp and mesocarp, where they colonize and degrade host cells through enzymatic activity. As infection progresses, white mycelial mats and green conidia develop on the fruit surface, which are characteristic of Green Mold symptoms. Under optimal conditions (around 25 °C), the disease cycle can be completed within 3–5 days, with each infected fruit generating up to one to two billion spores that are easily dispersed by air currents. Importantly, the pathogen can remain quiescent in apparently healthy fruits, making early detection before sporulation difficult.^[12].

Sour Rot

Sour Rot is one of the most significant post-harvest diseases in global citriculture, ranking just behind Blue Mold and Green Mold in frequency. It has been reported in nearly all citrus-producing regions and poses a serious challenge, particularly in injured or overripe fruits, as well as those stored for extended periods. Conditions such as prolonged rainfall and inadequate pre-or post-harvest management favor its occurrence^[7].

The yeast-like fungus Geotrichum candidum is the causal agent of Sour Rot and can be identified by different denominations, such as G. candidum var. citri-aurantii, Galactomyces citri-aurantii, or G. candidum citrus race, all associated with its ability to infect citrus fruits and grow in environments with pH below 2.72^[8].

It is important to recall that we chose to work with this disease after discussions with the orange-producing company AlfaCitrus. As a key stakeholder in our project, AlfaCitrus highlighted the urgent need to combat Sour Rot, making its control essential for reducing orange losses during post-harvest handling.

Disease Symptoms

In the early stages of Sour Rot, infected oranges develop light brown lesions. Subsequently, the peel becomes covered with a whitish mold and, due to the decomposing action of the fungus, it causes tissue breakdown, leading to juice leakage. The orange then starts to decay, releasing a putrid odor that is characteristic of this phytopathology (Figure 5)^[7].

Biological Cycle

Infection occurs through peel injuries, which allow the fungus to penetrate the albedo. Initially, the lesions appear water-soaked with a dark yellow coloration, later progressing to epidermal wrinkling and to the development of a white to cream-colored mycelium. The fruit eventually liquefies within a few days, forming a viscous mass often associated with Green Mold^[7].

Sour Rot can develop either before or after harvest and its incidence may be favored by the degreening process, such as high humidity and frequent rainfall. The presence of a water film on the fruit is essential for fungal penetration, and its dissemination occurs both through direct contact between infected and healthy fruits and through the use of contaminated boxes, bags, and containers. For its development, G. candidum requires relative humidity above 92% and temperatures above 10 °C, with the optimal range of 25 °C to 30 °C being the most favorable for rapid fruit degradation^[7].

Current Disease Management

Greening

Greening management involves integrated strategies aimed at controlling the vector, preventing the spread of infection, and minimizing the impact on already affected trees. The choice of the most appropriate measure to be applied depends directly on the level of infestation in the area. In regions where disease incidence is low, priority actions include the removal of symptomatic trees to prevent pathogen spread, protection of orchard edges through intensive monitoring, and the application of insecticides to control D. citri, the main vector of the disease. In addition to chemical control, the use of biological control agents has been employed to sustainably reduce vector insect populations^[1,2].

For already infected trees, management practices aim to mitigate damage and prolong plant lifespan. Key actions include nutritional supplementation through foliar sprays of rapidly absorbed nutrients and phytohormones, as well as soil pH adjustment to enhance nutrient uptake^[1,2].

Between 2019 and 2020, citrus growers in Florida began applying the antibiotics streptomycin and oxytetracycline to suppress the spread of Greening. To this day, this management practice continues in American citrus production. Nevertheless, there are no consistent studies ensuring the long-term safety of these compounds, both in terms of human health and environmental impact^[6].

Green Mold

The management of citrus plantations affected by Green Mold commonly involves the standard procedure of applying fungicides, especially imazalil and thiabendazole. However, studies have reported resistance of P. digitatum to these compounds, and there are also concerns regarding the application of these fungicides in relation to the impact to human health and the environment^[11].

In this context, alternative methods of disease control are currently being explored, such as the application of carbonates, chlorides, and natural extracts; hydrothermal treatment with a spore removal system; and exposure to ultraviolet radiation^[13]. Therefore, the search for efficient and innovative solutions to combat Green Mold is extremely necessary.

Sour Rot

As conditions favorable for fungal development are often present during the storage and transport of oranges, disease control becomes a major challenge. Although fungicides such as imazalil, thiabendazole, pyrimethanil, and fludioxonil are used to combat Green and Blue Molds, these products are not effective against Geotrichum candidum^[9].

New products have been studied internationally to fill this gap. In 2013, the European Union and the United States approved Melanite for the control of Sour Rot, but its efficacy has not yet been tested under Brazilian conditions. Another option, sodium ortho-phenylphenate (SOPP), can reduce disease severity; however, its use is limited due to fruit peel damage and human health concerns, as it is considered carcinogenic^[7].

Among the fungicides that could be applied for Sour Rot, guazatine is the only one with direct activity against Geotrichum candidum, representing one of the few disease management options. However, its use is prohibited in several countries, including Brazil, due to the lack of conclusive studies on its metabolism in citrus fruits and potential health risks. In light of the lack of registered control agents, the need for safe and effective alternatives becomes increasingly urgent^[7]. Figure 6 shows a summary of the possible management of citrus diseases.

Climate Change and its relation to citrus diseases

Climate change refers to long-term alterations in the planet’s climate and temperature patterns. These changes can occur naturally, due to the solar cycle, but are being strongly accelerated by anthropogenic activities, which are based on human modifications of the natural environment. The burning of fossil fuels and deforestation of natural vegetation are examples of anthropogenic activities that lead to intense greenhouse gas emissions. The release of these gases, especially carbon dioxide, contributes to the rise in global temperatures and, consequently, changes in climate patterns^[19].

Agriculture is among the sectors most vulnerable to these changes. In addition to being exposed to destructive natural phenomena, such as droughts, wildfires, and floods, studies indicate that alterations in climate patterns contribute to the widespread propagation of plant diseases among crops. Climate change can make crops more susceptible to infections by affecting pathogen development, altering host–pathogen interactions, changing vector physiology, promoting the emergence of new pathogenic strains, and weakening plant resistance to pathogens^[20].

Rising temperatures, irregular precipitation regimes, and increasing atmospheric CO₂ concentration have been reshaping the global citrus production landscape. These environmental shifts directly influence the physiology of citrus plants, reducing water availability, altering phenological cycles, and predisposing plants to infection. The vulnerability of citrus crops is also linked to their genetic homogeneity and perennial nature, which allow pathogens and vectors to persist in the same orchards for long periods. Under higher thermal and water stress, the natural defense responses of citrus plants decline, favoring opportunistic fungi and bacteria. Furthermore, the bibliometric analysis shows that most research on climate change and citriculture focuses on water management and disease dynamics, indicating a growing concern over the combined impact of abiotic and biotic stressors on crop health and sustainability^[21].

In Brazil, one of the most comprehensive assessments of how projected climate scenarios for 2020–2080 could affect major citrus diseases in São Paulo State revealed that climatic factors, particularly temperature, precipitation and humidity, play decisive roles in the epidemiology of these diseases. Among them, Candidatus Liberibacter asiaticus, the bacterium associated with huanglongbing (HLB or Greening), relies on the psyllid Diaphorina citri for transmission, and the population dynamics of this insect are strongly temperature-dependent. Rising average temperatures shorten its life cycle, enabling multiple generations per year, which may allow the disease to spread into areas previously considered climatically unsuitable^[22].

Beyond biological mechanisms, climate change also alters the economic and management dimensions of disease control. In this context, a recent study ^[23] developed a dynamic bioeconomic model that integrates climatic variability and disease management to determine optimal strategies for citrus farms affected by Witches’ Broom Disease of Lime (WBDL). The model revealed that under extreme climatic conditions, particularly in arid and semi-arid regions, disease severity significantly increases, reducing the overall economic value of the farm. However, adaptive management, including timely pruning, replanting, and intensified monitoring, can mitigate these losses. The study also showed that optimal intervention frequency and intensity vary by climatic zone, suggesting that flexible, climate-informed decisions can sustain or even improve farm profitability under harsher conditions. This framework can be applied to other citrus diseases, emphasizing that long-term sustainability depends on adaptive, evidence-based management.

Since climate change is an unavoidable reality, the adoption of alternative management strategies becomes crucial. Among these, the use of AMPs emerges as a promising and sustainable approach for controlling citrus phytopathologies. These biologically active molecules can disrupt pathogen membranes, offering an alternative to conventional chemical pesticides. Thus, our project aims to use a synthetic biology strategy for the large-scale production of AMP, specifically CTX, aimed at controlling the major pests and pathogens that threaten the citrus chain, including Greening (Candidatus Liberibacter spp.), Green Mold (Penicillium digitatum), and Sour Rot (Geotrichum candidum).

By developing AMP-based biocontrol solutions, we aim to reduce the dependency on agrochemicals, mitigate environmental contamination, and enhance food security — contributing to the United Nations Sustainable Development Goals (SDGs), particularly: SDG 2 – Zero Hunger, through the protection of crop productivity and food systems and SDG 12 – Responsible Consumption and Production, by encouraging biotechnological alternatives to harmful agrochemicals. Thus, our project bridges science, sustainability, and entrepreneurship, showing how synthetic biology can provide real-world solutions to one of the greatest challenges of modern agriculture: safeguarding citrus crops, and global food security in a changing climate.