**6. MVOCs in the management of postharvest diseases of fruits**

#### **6.1 VOCs produced by fungi**

The use of VOCs for the control of postharvest diseases of fruits is well represented by the report [77]. on an endophytic fungus, *Muscodor albus*, discovered in *C. zeylanicum* in a Honduras botanical garden. In the late 1990s, an isolate of *M*. *albus* was identified as a good fungal bioagent for the biofumigation of fruits and vegetables after harvest to control apple and peach decay [78], green mold and sour rot of lemon [79] and gray mold of table grapes [80]. The ability of *M. albus* to manage postharvest diseases depends on the medium that supports the growth of an endophytic fungus which in turn greatly influences the quality, emission and the effectiveness of VOCs. It has also been reported that more than 28 VOCs of five groups of organic compounds such as acids, alcohols, esters, and lipids was identified through GC/MS analysis from an endophytic fungus [37]. About 48.5 per cent of 2-methyl-1-butanol serving a major component along with second and third compound as isobutyric acid (14.9%) and ethyl propionate (9.63%) were identified from *M. albus* inoculated in an autoclaved rye [78].

Even though *Oxyporus latemarginatus* is a plant pathogen affecting trees, an isolate of this species EF06 that produced VOCs was isolated from the healthy tissues of pepper plants which acts as a potential biological agent [81]. *O. latemarginatus* EF06 tested in half-plate divided Petri plates produced VOCs and inhibited the mycelial growth of *Alternaria alternata*, *B. cinerea*, *C. gloeosporioides*, *Fusarium oxysporum* f. sp. *lycopersici*, and *R. solani*. The gaseous VOCs produced by *O. latemarginatus* EF069 multiplied in a wheat bran–rice hull cultures of 50 g upon exposure to apple fruits in closed container suppressed 98.4 per cent development of *Botrytis* lesions at 20°C. The hexane extract of wheat bran–rice hull cultures of *O. latemarginatus* EF069 was used for the identification of an antifungal VOCs by repeated silica gel column chromatography and identified as 5-pentyl-2-furaldehyde (PTF). The purified PTF were effective against various plant pathogens. The mycofumigation with EF069 was also effective in reducing postharvest decay of apple fruits caused by *B. cinerea* [82].

The most common fungal pathogen during storage conditions is *P. expansum* [83]. But there are few reports of antifungal VOCs extracted from this species. VOCs from *P. expansum* R82 have been used to control postharvest diseases of fruits. *P. expansum* R82 tested in double Petri dish assays was found to be able to inhibit mycelial growth of postharvest pathogens *viz*., *B. cinerea*, *Monilinia* spp., *C. acutatum* and other strains of *P. expansum* by producing VOCs [84]. The SPME-GC analysis revealed the presence of geosmin and phenethyl alcohol (PEA) as the major terpenoid and alcohol VOCs produced respectively by the *P. expansum* R82. Synthetic PEA does not show any inhibitory activity when tested *in vitro* against the pathogens. Without any direct contact of *P. expansum* to the fruits, the fungus could be grown in a separate chamber and the produced VOCs can be transferred to the storage room through pump and further can be exposed to fruits to control postharvest diseases [85].

The different VOCs produced by *Ceratocystis fimbriata* showed strong bioactivity against a wide range of pathogens including postharvest diseases such as peach brown rot and citrus green mold. The volatiles exposure of *C. fimbriata in vitro* to the peach and citrus fruits against *M. fructicola* and *P. digitatum* pathogens showed strong inhibition towards mycelial growth, conidial production and spore germination and the *in vivo* exposure lead to the reduction of disease over control of 92 and 97 per cent respectively. Exposure to VOCs of *C. fimbriata* lead to misshapen hyphae and conidia when observed under scanning electron microscopy also their pathogenicity was

*Volatile Organic Compounds Produced by Microbes in the Management of Postharvest Diseases… DOI: http://dx.doi.org/10.5772/intechopen.110493*

greatly reduced. The VOCs were identified as butyl acetate, ethyl acetate and ethanol by head space GC–MS [86].

In spite of the fact that the storage temperature may affect either VOC emission or control activity, storage at 20°C was effective compared to 5°C then period of exposure of VOCs can increase from 4 to 24 h, obtaining the same level of efficiency [79].

#### **6.2 VOCs produced by bacteria**

Different species of *Bacillus* and *Pseudomonas* have displayed inhibitory effect against the growth of postharvest pathogens of fruits with multiple modes of action, including the production of VOCs.

*Bacillus subtilis* strain CL2 showed antagonistic effect upon producing the VOCs *in vitro* `against wolfberry postharvest pathogens by inhibiting the hyphal growth of *Mucor circinelloides* LB1, *Fusarium arcuatisporum* LB5, *Alternaria iridiaustralis* LB7, and *Colletotrichum fioriniae* LB8 using two-sealed-base-plates method. The mycelial morphology of the inhibited pathogens were deformed, twisted, folded, and shrunken when observed under scanning electron microscope. The VOCs could also reduce the weight loss and decay incidence rate of wolfberry fruits infected by the postharvest pathogens. The headspace-gas chromatography-ion mobility spectrometry analysis, revealed the production of seven VOCs by strain CL2. Among them, 2,3-butanedione and 3-methylbutyric acid are the main antifungal active substances [87].

The volatilome produced by three strains of *Bacillus velezensis* (BUZ-14, I3 and I5) displayed inhibitory effect *in vitro* against *B. cinerea*, *M. fructicola*, *M. laxa*, *Penicillium italicum*, *P. digitatum* and *P. expansum.* Among three strains I3 and I5 showed 100 per cent inhibition of *B. cinerea*. The volatile metabolites of I3 also reduced 50 per cent inhibition of grapes gray mold and BUZ-14 decreased apricots brown rot severity by reducing the *M. fructicola* infection from 60 to 4 mm. The main volatiles responsible for showing its antifungal activity identified from SPME coupled with GC–MS ranged from 12 to 15 compounds including 2-nonanone, 2-undecanone, 2-heptanone, 1-butanol, acetoin, benzaldehyde, butyl formate, diacetyl, nonane, or pyrazine, benzaldehyde and diacetyl. Among those VOCs diacetyl was able to control 60 per cent infection of gray mold in table grapes and blue rot in mandarins with only 0.02 mL L−1 concentration*.* The diacetyl and benzaldehyde VOCs have been identified as promising compounds and can be applied in active packaging during the postharvest storage, transit and trade of fruit crops. However, prior to the application of any VOCs, it is crucial to determine the active dose as well as the phytotoxicity of the volatiles, since some of the fruits such as apricots and apples have proven to be highly sensitive [88].

*In planta* prophylactic fumigation of mango fruits cv. *Amrapali* with 24 h exposure of either endophytic bacteria *Pseudomonas putida* BP25 or the identified volatile from the bacteria i.e., synthetic VOC 2- ethyl-5-methylpyrazine at 25°C showed a reduction of more than 76 per cent of anthracnose severity on fruit. Additionally, the physicochemical properties of fumigated fruits were also increased representing a new compound for the postharvest management of mango during its commercialization [51].

The downy blight of litchi caused by the oomycete pathogen *P. litchii* severely suppress the production and quality of litchi fruit. Fumigation of litchi fruits with *B. amyloliquefaciens* PP19, *Exiguobacterium acetylicum* SI17 and *B. pumilus* PI26 volatiles reduced the disease severity of downy blight. The volatile profiles identified from the above-mentioned bacterial strains *viz*., 1-(2-Aminophenyl) ethenone, benzothiazole and α-farnesene displayed inhibitory effect against the downy blight and serves as promising VOC for the postharvest diseases control of litchi fruits [89].

The VOCs produced from *B. thuringiensis* and *B. pumilus* decreased the mango anthracnose infections by 88.5 per cent [90]. When VOCs emitted from *B. subtilis* and *B. amyloliquefaciens* were assayed alone or in combination for their antifungal property against *Penicillium* infection on citrus, the volatile profiles of *B. amyloliquefaciens* (8 VOCs) and *B. subtilis* (21 VOCs) reduced the disease incidence of citrus by *P. crustosum*, but no synergetic effect was exhibited in citrus fruit treated with the antagonist combination. The volatile profile included N-containing compounds, alcohols and ketones were common in both of them. There were morphological abnormalities in *P. crustosum* when exposed to VOCs. There was a swelling in the hyphae, sporangium and conidia [91].

Upon exposure of VOCs produced by the *B. subtilis*, there was a retraction of protoplasm with in the hyphal tips and separation of an empty hyphal segments in germinating conidia of *B. cinerea*. In addition, due to the protoplasm retraction pathogen conidia could not germinate, even after the withdrawal of VOCs, indicating that the protoplasm damage may be irreversible and lethal for pathogens [28].

*Streptomyces* spp. is group of actinomycete capable of reducing the growth of certain fungal pathogens such as citrus *P. italicum* [33], gray mold of tomato fruit [92] and strawberries [32] caused by *B. cinerea* due to the ability of emitting VOCs. Volatiles of *S. platensis* F-1 displayed a strong reduction in strawberry gray mold incidence by 73 per cent. Among 16 volatiles identified from *S. platenesis* F-1, VOCs phenylethyl alcohol and (+)-epi-bicycle sesquiphellandrene were previously been detected in *M. albus* [37] and in the *Kleina odora* essential oil [93] respectively. Patel *et al.* (Unpublished data) reported that acetic acid 2-phenylether ester, styrene, β-Phyllandrene and thujopsene were most abundant VOCs released from *B. amyloliquefaciens* against postharvest pathogens of grapes.

#### **6.3 VOCs produced by yeast**

Yeasts as bioagents have been extensively studied since they own many features that satisfy the requirements for being biocontrol agents in fruits [94, 95]. Yeast species usually require a simple nutritional diet, colonizes on dry surfaces for longer periods and can grow rapidly on less expensive substrates in bioreactors [96]. Importantly, they do not produce any kind of allergenic spores or mycotoxins as many fungi or antibiotics which might be produced by bacterial antagonists [97, 98]. In addition, yeasts are a major constituent of the epiphytic microbial community of fruits and vegetables and also phenotypically adapted to this niche.

*Candida intermedia* 410 inhibited incidence of strawberry gray mold by the production of volatiles. It has been confirmed that VOCs production were the only probable mechanism against *B. cinerera* because there was no direct contact between the pathogen and yeast cells. The most abundant compounds identified from *C. intermedia* were 1,3,5,7-cyclooctatetraene, 3- methyl-1-butanol, 2-nonanone, and phenethyl alcohol and confirmed that yeast can be potentially developed as a biofumigant for the control of gray mold of strawberries [99].

Strains belonging to different species of yeasts such as *Saccharomyces cerevisiae*, *Wickerhamomyces anomalus* and *Metschnikowia pulcherrima* showed both *in vitro* and *in vivo* inhibitory effect due to the emission of VOCs on *B. cinerea* causing postharvest bunch rot of table grapes [100].

The VOCs produced by *S. cerevisiae* inhibited *Phyllosticta citricarpa*, causing black spot of citrus. Individual exposure of 3-methyl-1-butanol and 2-methyl-1-butanol controlled the development of new lesions close to 90%, even after removing the

*Volatile Organic Compounds Produced by Microbes in the Management of Postharvest Diseases… DOI: http://dx.doi.org/10.5772/intechopen.110493*

fruits from the VOC influence and displayed effective inhibition of mycelial growth, appressorium formation and germination of conidia [101].

The psychrotrophic, non-pectinolytic yeast *Candida sake* grown in apple juice act as a potential biocontrol agent. The antifungal volatile organic compounds produced by *C. sake* inhibited the growth of five postharvest pathogens of apple (*P. expansum*, *B. cinerea*, *A. alternata*, *A. tenuissima* and *A. arborescens*). The VOCs were also effective on *in vivo* assays to control *P. expansum* in Red Delicious apples [102]. Patel *et al.* (Unpublished data) reported that acetic acid 2-phenylether ester, styrene, β-Phyllandrene and thujopsene were most abundant VOCs released *Hanseniaspora opuntiae* against postharvest pathogens of grapes.

## **7. Safety of microbial volatile organic compounds**

The disease control using VOCs from microbes would be safer to human health and environment as the yeast and their products are Generally Recognized As Safe (GRAS) by the U.S. Food and Drug Administration (FDA), and the yeast is classified as Biosafety Level 1 by U.S. Centres of Disease Control and Prevention (CDC/OHS, 2009), as it is not a human pathogen, it generally does not produce mycotoxins, antibiotics, or other molecules that are unacceptable in foods [100]. Many antimicrobial VOCs, such as decyl alcohol, nonanal, acetoin and phenylethyl alcohol, are already in use as additives in foods and cosmetics and their use can be extended to control postharvest diseases in fruits and vegetables [103]. However, they are reports on the issues related to safety of these mVOCs to human being. For example some mVOCs reported as allergenic and asthmatic agents such as 1-octen-3-ol [104]. Many of the reports arise from analysis of environmental samples from moist and damp rooms or closed places. As mentioned by Piechulla and Degenhardt [105] the use of these compounds in post-harvest disease management depends on their characterization, dose and their mode of action. These mVOCs need to be applied in very low concentration and they are completely degradable. Compared to synthetic fungicides, they are less harmful due to no residual toxicity. To harness the use of mVOCs, a prerequisite is the availability of adequate in vitro test systems to generate the data to facilitate the legal and regulatory authorities in giving permission for their use in agriculture. A review by Ceremi et al. [106] give the list of in vitro systems for the evaluation of mVOCs on human being. They reviewed the submerged cultivation, air-liquid-interface (ALI), spheroids and organoids as well as their advantages and disadvantages. As these mVOCs are suspected to be allergenic, the methods to study the effects on human respiratory tract need to be updated. In our work too, (Pooja et al. unpublished data) though styrene was abundantly produced by *Hanseniaspora opuntiae* and *B. amyloliquefaciens*, but we did not use it due its ill effect on human.

#### **8. Conclusion**

Microbes are a remarkable source of active chemicals due to their diverse chemical makeup. VOCs in particular, when compared to traditional products, can offer evident environmental benefits due to their nil residual effect, renewability, biodegradability, and low toxicity. This makes them an effective aspect of an eco-chemical approach in the management of postharvest diseases. As the world is moving towards a green economy, with new production chains that begin in agriculture and end by

returning back to agriculture. Products and by-products come together to establish a sustainable economic system that uses renewable resources. Locally and in many agricultural exporting nations, laws to limit chemicals are currently being adopted. In order to counteract the negative effects of microorganism infection during storage, these non-toxic and GRAS-recognized compounds will be added to the postharvest chain. This will improve the protection of human health and the environment. The use of mVOCs in disease management is evolving and its beneficial effect without harming human health need demonstration after in depth molecular studies to confirm their potential use in agriculture. The future thrust areas as suggested by Kanchiswamy [54] include application of nanotechnology in delivering these mVOCs, expanding the database of mVOCs, studies on mode of action of individual compounds and synergistic effect of cocktail of compounds, non-volatile biodegradable precursors of mVOCs and molecular basis of their mode of action.
