Biological control strategy for postharvest diseases of citrus, apples, grapes and strawberries fruits and application in Indonesia

In Indonesia, the postharvest fruit loss is 25%, so the economic loss from the export of various fruits is estimated at US$ 58,966,861. One of the causes for the loss is postharvest pathogens. Postharvest fruit rot is caused mainly by fungi, some of which produce mycotoxins harmful to human health. Therefore, in meeting the global food safety requirements, Indonesia should develop a biological control strategy for postharvest fruit diseases. This paper is a review based on observations, a literature review of postharvest biological control of citrus and other subtropical fruits, and an overview of strategies and prospects for their application in Indonesia. The pathogens that cause diseases on citrus fruits, apples, grapes, and strawberries in Indonesia produce mycotoxins, namely Fusarium sp., Aspergillus terreus, Aspergillus sp., Penicillium sp., and Alternaria sp. The potential biological agents are from the yeast group, such as the Candida genera, the bacterial group, such as the Bacillus and Pseudomonas genera, and the fungal group, such as the Muscodor and Trichoderma genera. Through mutually additive and synergistic multiple reduction methods in cooperation with the vanguards, postharvest disease control emphasizes disease prevention using several methods. Each method reduces the percentage of damage by a certain amount to produce highly effective controls.


Background
Indonesia is one of the tropical countries with the potential to develop fruits as a commodity, including citrus and other subtropical fruits (apples, grapes, and strawberries). The problem of fresh product quality and safety still needs attention. The low quality of fruits is mainly caused by a high microbial contamination, which pollutes various fruits in Indonesia because they are supported by tropical climatic conditions with high humidity levels. This contamination is economically detrimental and harmful to health, especially for diseases caused by fungi that produce mycotoxins. Mycotoxins are toxic organic compounds produced by the secondary metabolism of fungi that can interfere with human health clinically and pathologically (Ismaiel and Papenbrock 2015).
Pathogens that infect fruit products, including citrus, grapes, apples, and strawberries, may come from the field, while some emerge during postharvest. In addition, inadequate postharvest handling of fruits can result in physical damage, for example, bruising because of falling during transportation. Bruises or other physical damages on fruits make them easily infected with fungi. Postharvest and food losses are around 30% of production in Fungi, bacteria, and viruses can cause postharvest diseases of citrus, apples, grapes, and strawberries. Postharvest diseases can occur when the commodity has been harvested, but infections may also happen before harvesting when the pathogen is in a dormant state. Pathogens then develop and cause symptoms when conditions are favorable. Inventory-specific research showing postharvest diseases of citrus, apples, grapes, and strawberries has not been widely carried out in Indonesia. However, from the results of literature reviews, field and market observations (Dwiastuti and Fajrin 2014;Widiastuti et al. 2015;Dwiastuti et al. 2016), and during the authors' visit to production centers of citrus, apples, grapes, and strawberries at different times, infections of Penicillium italicum, P. digitatum, Lasiodiplodia theobromae, Alternaria citri, Phomopsis citri, Geotrichum candidum, Pezicula malicorticis (Gloeosporium malicorticis), Glomerella cingulata, Physalospora abtusa, Podosphaera leucotricha, Mucor piriformis, P. expansum, Botrytis cinerea, Erysiphe necator, Colletotrichum gloesporiodes, Rhizopus stolonifer, Aspergillus sp., Penicillium sp., Phytophthora cactorum, Fusarium sp., Alternaria sp. were found in traditional markets and supermarkets ( Table 1).
The types of fruits observed varied, from local to imported fruit varieties. More fungal contaminations were found in traditional markets than supermarkets. Lower fungal contaminations of fruits in supermarkets happen since supermarkets have air-conditioned storage areas, and fruits are sorted, cleaned, and covered with wrappers, reducing contact among fruits. In traditional markets, fruits are sold or stored as a reserve, placed in bamboo baskets without cooling space, while the temperature in the market tends to be high. In addition, fruits in traditional markets are not individually wrapped, so contact among fruit is possibly very high.
Some fungal diseases that contaminate citrus, apples, grapes, and strawberries in Indonesia including Fusarium sp., A. terreus, Aspergillus sp., Penicillium sp., and Alternaria sp. The types of mycotoxins found were patulin, ochratoxin, aflatoxin, deoxynivalenol, and others ( Table 2). Each of these mycotoxins can cause quite dangerous effects on humans and animals. Table 2 shows that mycotoxins can cause mild to severe symptoms, including moderate-acute nausea, vomiting, diarrhea, abdominal pain, headaches, dizziness, fever, respiratory and circulatory failure, paralysis, seizures and death, to carcinogenic and mutagenic effects, plasma membrane disruption, protein synthesis inhibition, and inhibition on the anti-tumor, antiviral, antibacterial activities. However, in-depth knowledge about mycotoxins, especially in fruits in Indonesia, is still limited.

Control management
Similar to pre-harvest disease management in the field, postharvest disease control must also consider an integrated pest management strategy. Postharvest handling alone is insufficient for the product after harvest, but control measures during pre-harvest will significantly determine the health quality of the harvested fruits. Postharvest disease control is highly dependent on the initial condition of the commodity, meaning that plant health, planting area cleanliness, and several other treatments in the field determine the presence or absence of postharvest diseases. In addition, after entering the postharvest Page 3 of 12 Dwiastuti et al. Egypt J Biol Pest Control (2021) 31:141 period, disease progression is highly dependent on management techniques from harvesting to transportation or storage at the consumer level. Disease control for citrus, apples, grapes, and strawberries is usually carried out from generative growth and ends one month before harvest to avoid residue on the fruits (Sumiati and Julianto 2017). Generally, fruit orchards in Indonesia are located far from consumers. Therefore, fruits are transported from the packaging station in the production center to various destinations using various vehicles. They can even be transported several times to reach the final destination (Prabawati 2009). This causes farmers to sell on a wholesale basis. As such, sorting is not carried out by farmers but by collectors, traditional market traders, and retail traders by removing rotten fruits in simple to modern ways.
However, not all farmers or traditional traders in Indonesia carry out postharvest handling, such as washing,  Dwiastuti et al. Egypt J Biol Pest Control (2021) 31:141 sorting, and pre-cooling (Siswadi 2007). As a result, many of them are not aware of whether the fruit carries spores or parts of microbes that can develop and become new sources of infection. In addition, most of the local market traders (traditional markets) in Indonesia only select fruits based on their respective grades and put them in the same container; there is no separation among fruits. The exception is only for packaging for modern markets (supermarkets), which require more stringent terms and standards for the safety of fruit to be marketed (Siswadi 2007). Postharvest disease control using physical method has been recommended in Indonesia, but not all can apply it to maintain fruits quality. The method used includes low-temperature management and modification of the storage room atmosphere using reduced oxygen, elevated carbon dioxide, heat treatment (hot air or immersion), or radiation (Sukasih and Setyadjit 2019). Utilizing chemicals to control the postharvest disease is considered the most effective. However, its application depends on the type of marketing and infection that occurs. For example, in citrus with a long shelf life, there should be additional safe chemical treatment to prevent the germination of fungal spores. Conversely, for strawberries that have a very short shelf life, it seems that postharvest disease control treatment is prioritized on controlling just before harvest in the field (Sudjatha and Wisaniyasa 2017).
Alternative modern techniques used to minimize losses due to postharvest diseases in developed countries are physical, biochemical, biological (Qadri et al. 2020), and breeding for resistance (Pétriacq et al. 2018). These modern techniques can be used as a reference for controlling postharvest diseases in Indonesia. Until now, there is no information on citrus, apple, grape, and strawberry varieties that are resistant to postharvest diseases.
Biochemical techniques for modern postharvest disease control are aimed at suppressing the use of pesticides and minimizing the side effects of fungicide resistance in major fungal pathogens such as Penicillium, Monilinia, Botrytis, and other common pathogens (Qadri et al. 2020). The technique being tried out involves genetic transformation with the application of fungicidal dsRNA. de Oliveira Filho et al. (2021) reviewed the results of research on the external application of synthetic fungicide dsRNA on fruits, which is safer for controlling postharvest fruit diseases, as a new generation of sustainable and environmentally friendly products. Several studies have been carried out by spraying or soaking fruit surfaces with RNA interference (RNAi) to control postharvest fungi of fruits, particularly B. cinerea (Wang et al. 2016) and Fusarium spp. (Gu et al. 2019) which causes postharvest losses on strawberries, grapes, and other fruits. This new fungicide with RNAi technology has greater specificity and efficiency. External application of double-stranded RNA (dsRNA), small interference RNA (siRNA), or hairpin RNA (hpRNA), resulting in post-transcriptional gene silencing, has emerged as a promising strategy for controlling phytopathogens (Islam and Sherif 2020). Nanomaterials have also been used as a new generation in postharvest technology in general and disease management in particular (Roberto et al. 2019). Exogenous application of calcium-based chemicals helps In addition, Youssef et al. (2020) reported that potassium bicarbonate (PB), sodium silicate solution (SSi) and calcium chelate (CCh) (1%) were effective in inducing resistance to gray mold disease in table grapes and reduced disease incidence by 43, 50, and 41%, respectively. The salt solution acts as an antimicrobial agent against the gray mold of table grapes so that it can increase the natural host resistance associated with an increase in the antioxidant system. From this study, it was proved that the activity of the enzymes superoxide dismutase (SOD), ascorbate peroxidase (APX) and peroxidase (POD) increased with the above salt treatment. Various physical techniques with refrigeration, cold atmosphere storage, low pressure storage, and modified atmosphere storage are used to control postharvest disease. The physical technique of heat treatment is considered the most effective, especially to treat fungal diseases and chilling injury. In addition, UV treatments (UV-C, UV-B, and UV-A) are used to sterilize commodities, reduce spoilage due to microorganisms, extend shelf life, and maintain fruit quality. UV treatment exposes fruit and vegetable commodities for a period of time under a UV lamp bank, with maximum emission at 254 nm. UV-C treatment has been shown to be effective for controlling the most common postharvest pathogens (Civello et al. 2006). Several studies have also proven that UV-C radiation is effective not only because of its disinfecting effect, but also because it can stimulate plant defenses. Forges et al. (2020) stated that UV-C radiation was efficient in reducing disease progression in many species, including strawberries (Fragaria × ananassa). Infection on leaf against powdery mildew and on fruit against Rhizopus spp. were greatly decreased due to the UV-C effect. Besides that, it also has an impact on the initiation of flowering and fruit development, increasing the physical integrity of strawberries in storage, although it does not increase the shelf life of strawberries.
Postharvest disease management using biological agents has been studied extensively. By involving natural enemies of the pathogen to be targeted, the population of the pathogen can be suppressed through restriction of normal growth or activity. In addition, volatile compounds are usually applied on a commercial scale for flavoring and seasoning in foods, which greatly reduces the incidence of microbial pathogens. This volatile compound has various properties such as antiprotectant, antimicrobial, less harmful to mammals, environmentally friendly, and can be used as an alternative to chemical fungicides (Abo-Elyousr et al. 2021). Plants are a large reservoir of natural compounds that harbor fungicidal activity with the potential to replace synthetic fungicides. Many species produce volatile substances and essential oils that can serve as antifungal or antimicrobial preservatives for fruits and other harvested commodities. Thus, combining multiple treatment options can offer stakeholders and producers a more consistent, durable, practical, and sustainable solution for postharvest disease control.
The breeding techniques involved in increasing fruit resistance and postharvest disease control is priming. This is a novel adaptive strategy by inducing plant immune systems to have protection against disease (Pétriacq et al. 2018). Priming can be long lasting and can be maintained throughout the plant life cycle (Luna et al. 2016) and can even be transmitted to the next generation (Rasmann et al. 2012), therefore representing a type of plant immunological memory. This phenomenon is characterized by increased activation of induced defense mechanisms. Stimuli can be from pathogens, beneficial microbes, or arthropods, as well as chemical and abiotic cues, thus triggering priming formation that acts as a warning signal. After perception of a stimulus, changes can occur in plants at physiological, transcriptional, metabolic, and epigenetic levels. This phase is called the priming of defense phase and is understood as the adaptive part of induced resistance (IR) (Mauch-Mani et al. 2017). After subsequent challenges, plants effectively mount a faster and/or stronger defense response that defines a postchallenge prime and results in increased resistance and/or stress tolerance. The potential for future use is very high and inexpensive; thus, detailed research support is needed.

Alternative biological control
Antagonistic microorganisms have been found to suppress the development of disease on the surface of the fruits. Controlling postharvest diseases of citrus fruit using antagonistic microorganisms is carried out in two ways: (a) using natural epiphytic antagonists that are already on the surface of the fruit; and (b) artificial introduction of selective microbial antagonists that control postharvest diseases. There have been many studies showing that antagonistic microorganisms are effective in controlling postharvest disease of citrus, apples, grapes, and strawberries, with various application methods (Table 3). In addition, some antagonistic microbial products have been developed from actively infected fruits. Until now, biological control has not completely replaced the use of fungicides or other chemicals in postharvest disease control. In the future, it is hoped that biological control can become more established as part of an  Page 7 of 12 Dwiastuti et al. Egypt J Biol Pest Control (2021) 31:141 integrated program of controlling major postharvest diseases caused by fungi.

Yeast as a biocontrol agent
Yeast is one of the best and safe alternative ingredients for the biological control of citrus, apples, grapes, and strawberries. The mechanism of action of yeast in postharvest disease inhibition includes competition for nutrients and the ability to form biofilms and colonize wounds (Parafati et al. 2016). Furthermore, yeast can produce extracellular polysaccharides, increasing its resistance and limiting the growth of pathogenic propagules. In addition, yeast can use nutrients quickly and reproduce at high speed (Sharma et al. 2009). Yeast can also tolerate common pesticides such as imazalil, tiabendazole, pyrimethanil, or fludioxonil (Perez et al. 2017). Several types of yeast were successful in controlling citrus fruit rot disease, namely C. saitoana, C. famata, C. oleophila, C. sake, C.  (Platania et al. 2012). Results of the latest invention showed that several yeasts, including Meyerozyma caribbica, have the potential as a promising biocontrol gene for the prevention of anthracnose (C. gloeosporioides) in passion fruit (Navarro-Herrera and Ortíz-Moreno 2020). Abo-Elyousr et al. (2021) reported that S. vanrijiae was also effective to control P. digitatum on lemon. Inhibition of the conidia of M. fructigena from apples in vitro was demonstrated by S. vanrijiae, G. geotrichum, and Pichia kudriavzevii from 67.6 to 89.2% and significantly reduced the incidence of disease, respectively, up to 84.02-89.5, 80,1-86.9, 56.3-86.9% than the control (Madbouly et al. 2020). However, despite many reports regarding yeast as a biological control agent, the number of commercial products available is still limited in Indonesia.

Bacteria as a biocontrol agent
The use of bacteria for postharvest disease control of fruits is one step ahead. Its products are available as biofungicides. It is easy to obtain bacteria as control material because they are available abundantly in nature. In most plant species, rhizosphere bacteria can be isolated from the surface of plant tissues, soil, roots, and various plants of the rhizosphere. In addition, there are endophytic bacteria in demand as biocontrol agents because they can colonize plant tissues to form internal defenses against pathogens (Liu et al. 2013). Antagonistic bacteria are well known for producing substances with antifungal and antibacterial properties (Yánez-Mendizábal et al. 2011). Bacillus and Pseudomonas genera are effective antagonists in controlling pathogens because they contain the chitinase enzyme that can hydrolyze the ß-1,4 bonds among N-acetylglucosamine (NacGlc) in chitin; this polysaccharide polymer makes up the cell walls of several pathogenic fungi and the exoskeleton of invertebrates. Therefore, chitinase is well known for its antifungal or microbial properties (Yu et al. 2008). Mohammadi et al. (2017) reported the isolation of extracellular enzymatic activity of potential bacteria (B. subtilis, B. pumilus, B. megaterium, and Agrobacterium radiobacter) to control blue mold diseases in citrus fruit (P. digitatum). Abd-El-Kareem et al. (2021) also reported the effectiveness of B. pumilus on strawberry black rot disease (Rhizoctonia solani, F. solani, and Phytium sp.). The results showed that the chitinase, glucanase, and protease enzyme reacted positively to control pathogenic fungi, except for the bacteria A. radiobacter. The inhibitory mechanism of bacteria is classified as antibiosis. Some of the antibiotics produced by antagonistic bacteria are iturin, an antifungal produced by B. subtilis, and pyrrolnitrin produced by P. cepacia (Layton et al. 2011). The process of antibiosis can also occur through the production of low molecular weight compounds produced by antagonistic bacteria, called volatile organic compounds (VOCs). This compound is active in low concentrations in several chemicals, such as alcohols, aldehydes, ketones, esters, lactones, terpenes, and sulfur compounds. Due to their volatility, these compounds can safely be transported long distances in heterogeneous environments in the form of solid, liquid, or gaseous compounds. He et al. (2020) reported that VOCs produced by B. methylotrophicus BCN2 and B. thuringiensis BCN10 were effective in controlling and suppressing the mycelium of five postharvest pathogens isolated from loquat fruits, namely F. oxysporum, Botryosphaeria sp., T. atroviride, C. oriumes, and P. expansum based on in vitro and in vivo experiments. The final results showed that the loquats remained fresh for ten days significantly. Significant progress was also found in the control of postharvest citrus diseases with the use of antagonistic bacteria such as B. subtilis, B. amyloliquefaciens, P. fluorescens, B. thuringiensis, B. licheniformis, B. pumilus, B. cepacia, B. gladioli, B. glathei, B. diminuta, E. aerogenes, E. cloacae, P. agglomerans, P. aeruginosa, P. corrugata, P. putid, P. syringae, and S. plymuthica (Table 2). P. fluorescens produces secondary metabolites products include: phenazine-1-carboxylic acid and 2,4-diacetylphloroglucinol, polysaccharides, and many others (Soesanto 2009).
Currently, there are 3 commercial Pseudomonas formulations are listed in the USA: Bio-Save 10 LP, Bio-Save 11 LP, and BlightBan A506 (Stockwell and Stack 2007). Bio-Save 10 LP and Bio-Save 11 LP contain the P. syringae strains ESC-10 and ESC-11, respectively. This product is applied in packaging stations to prevent postharvest fungal diseases on oranges, pome, stone fruit, and potatoes during storage. BlightBan A506 contains P. fluorescens strain A506 and is mainly applied to pear and apple trees during bloom to suppress bacterial blight. This biocontrol product can inhibit fungal diseases with moderate to very good criteria. In addition, the procedure is relatively easy to implement and can be integrated with conventional products for disease control. In 2020, it was reported that Streptomyces sp. CACIS-1.5CA was effective against the pathogenic fungi Colletotrichum sp., Alternaria sp., Aspergillus sp., Botrytis sp., Rhizoctonia sp., and Rhizopus sp. with percentages ranging from 30 to 63% (Evangelista-Martínez et al. 2020).

Fungi as a biocontrol agent
Utilization of antagonistic fungi to control postharvest diseases is less developed than those of yeast and bacteria. Antagonistic fungi show a broad spectrum of disease control but have volatile antimicrobial compounds (Mercier et al. 2007). Six antagonistic fungi (V. lecanii, Page 9 of 12 Dwiastuti et al. Egypt J Biol Pest Control (2021) Table 2). The fungi biofumigant M. albus, which produces specific low molecular weight volatiles, has been used to fumigate lemons in storehouses and has successfully controlled green fungal pathogens and acid rot (Mercier and Smilanick 2005). This fungus produces 28 volatile organic compounds that exhibit some inhibitory effects against fungal and bacterial pathogens (Strobel et al. 2001). This antagonism occurs due to antimicrobial metabolites (Verma et al. 2007). The potential as fungal antagonists can be increased continuously with improved isolation, formulation, and application methods, particularly in the postharvest environment.

Strategy of implementing biological control for postharvest diseases in Indonesia
The application of biological control in postharvest diseases of citrus, apples, grapes, and strawberries has not been studied on a large scale. In traditional Indonesian markets, biological control is rarely implemented. Biological control alone is not quite popular and less desirable in Indonesia because their effectiveness as inhibitors are lower than synthetic pesticides. The biological control is also biodegradable and must be applied repeatedly. The main objective of developing a postharvest strategy is to ensure fruit products are safe and present the lowest possible risk. Therefore, a multi-method was applied in the supply chain from harvest to market to prevent cross-contamination and the spread of pathogenic contamination to the downstream process. This method was based on intervention using technology at critical points during the harvesting, handling, storage, shipping, and marketing processes, by integrating a field-to-supermarket approach. The concept of postharvest disease control was about using several methods, each of which reduces the percentage of damage by a certain amount, mutually additive and synergistic to produce control with 97-99% effectiveness (Fig. 1).
The following is a strategy for implementing postharvest disease biological control in Indonesia. Through mutually additive and synergistic multiple reduction methods in cooperation with the vanguards, postharvest disease control emphasizes disease prevention using several methods. Each method reduces the percentage of damage by a certain amount to produce highly effective controls carried out sequentially or together (Fig. 2). This multi-step method is divided into preventive and curative controls. Preventive control strategies are as follows: Some of the problems in the supply chain include the long distance between orchards and customers and the hilly and muddy, especially when it rains. Apart from being muddy, steep roads are also a problem to reach locations. Therefore, collaboration with local governments or ministries such as the Ministry of Agriculture, Action 1 = reducing the inoculum level through sanitation and/or careful harvesting and handling Action 2 = disinfectant treatment Action 3 = application of microbial antagonists Action 4 = cold storage, modified or controlled atmosphere storage or packaging Fig. 1 Overview of assumption of strategy effectiveness of postharvest diseases control for citrus and subtropical fruits in combination and synergy Page 10 of 12 Dwiastuti et al. Egypt J Biol Pest Control (2021) 31:141 of Industry, of Trade and Cooperatives, and of Tourism as market owners is highly recommended. In addition, fruit-picking tourism needs to be increased by promotion, especially before the main harvest season. The strategy that can be taken to keep strawberries fresh is to handle them immediately without delay after they are harvested.

Conclusions
Among postharvest diseases of citrus and subtropical fruit (apples, grapes, strawberries) found in Indonesia, some contain mycotoxins that are harmful to human health, including Fusarium, Aspergillus sp., A. terreus, Penicillium sp., and Alternaria sp. The biological agents in the yeast group (Candida spp.) have the opportunity to be a powerful postharvest disease controller. It has been developed abroad and can be synergized with the fungal groups (Trichoderma spp.) and antagonists bacteria (Bacillus spp. and P. fluorescens) found in Indonesia that are stable and have a broad spectrum. The potential botanical agents found in Indonesia are easy to make, have a broad spectrum, and can be combined with certain biological agents and chemicals. Through mutually additive and synergistic multiple reduction methods in cooperation with the vanguards, postharvest disease control emphasizes disease prevention using several methods. Each method reduces the percentage of damage by a certain amount to produce highly effective controls.