Postharvest Rot Research Articles

Utilization of the antagonistic yeast, Wickerhamomyces anomalus, combined with UV-C to manage postharvest rot of potato tubers caused by Alternaria tenuissima

Postharvest rot of potato tubers caused by fungal pathogens is the main cause of significant economic losses, while also raising potential food safety issues. Integrated disease management, utilizing bio-safe and eco-friendly methods, represents a sustainable strategy for reducing postharvest losses in crops, including potato. In the current study, the application of the antagonistic yeast, Wickerhamomyces anomalus, combined with a UV-C treatment was evaluated for the management of postharvest Alternaria rot of potato tubers, caused by Alternaria tenuissima. Both W. anomalus and UV-C as individual treatments reduced the size of A. tenuissima infections on potato tubers, relative to the control, while the combined treatment of W. anomalus and UV-C exhibited the highest level of inhibition. W. anomalus or UV-C alone, and especially when used in combination, induced the expression of defense-related genes, including polyphenol oxidase, peroxidase, and β-1,3-glucanase, and also increased the level of flavonoids and lignin in potato tubers. Our findings indicate that the mechanism of action by which UV-C enhances the biocontrol effect of W. anomalus against postharvest Alternaria rot includes the activation of defense-related response in potato tubers. The integration of biocontrol agents and physical treatments (e.g., UV-C) represents an effective, eco-friendly hurdle technology for managing postharvest rot in potato.

First report of Pythium aphanidermatum causing fruit rot of Cucumis anguria

Pythium aphanidermatum is reported for the first-time causing postharvest rot (cottony leak) on Cucumis anguria fruits (Cucurbitaceae). The fungus was identified based on morphological and molecular features and its pathogenicity was demonstrated. This is the first-time that this pathogen is reported on C. anguria in the world.

Diplodia bulgarica, cause of postharvest rot on apples in Turkey

Apple fruit cv. Golden Delicious symptoms of rot were collected from storage in the area of Eğirdir district of Isparta province, in Turkey. Based on morphology and phylogeny of DNA sequence data for the internal transcribed spacer (ITS) rDNA region, the isolated pathogen was identified as Diplodia bulgarica. This is the first description of D. bulgarica causing postharvest fruit rot on Malus domestica cv. Golden Delicious in Turkey and worldwide.

First Report of Stemphylium eturmiunum Causing Leaf Spot and Black Spot on Apple in Zhaotong, Yunnan, China.

Apple is the largest fruit tree crop in the world, and China is the largest apple-producing County in the world. Zhaotong, Yunnan Province is a typical cold and mountainous apple-producing area in China. However, apple production is threatened by diseases during the entire growing season, and among them, apple leaf spot and fruit black spot are severe. In previous reports, the main pathogen causing apple leaf spot and fruit black spot was Alternaria sp. (Lior, et al, 2017), while different pathogens were identified. In the current study, seven red Fuji apple fruit with typical black spot samples were collected randomly in Dongda company orchard, Sujiayuan town, Zhaotong, Yunnan on March 25, 2021. The spots on the surface of these apples appear rounded, the diseased parts turn brown or black in colour and the flesh became soften and rotten. The tissues of fruit epidermis at the edge between diseased and healthy parts were cut, soaked in 75% alcohol for 30 s, washed with sterile water three times, and air-dried. Five pieces of tissue were placed on PDA medium amended with rifampicin (50 mg/ml) and incubated in the dark at 25 ℃ for 3-5 days. After colonies grew, mycelial clumps were picked out from the edges of the colonies, transferred to new PDA plates, and incubated at 25 ℃ for 6 days. The diameter of the colonies reached up to 5.7 cm. A representative isolate was retained for further work and was named P6-3-1. The hyphae were white and dense at an early stage, the culture medium on the underside became yellow and the middle parts of the colonies were darker. With maturity, hyphae were clumped, became red with other colors interspersed, and the medium became dark red. Light brown spores were produced, with more vertical septa and fewer transverse septa. Two to three transverse septa were generally observed with obvious constriction at the transverse septa. Average spore size was 22.83 µm ± 2.04 µm × 14.58 µm ± 1.97 µm. DNA was extracted from mycelium, purified and amplified with two pairs of primers, ITS1/ITS4 (White et al. 1990) and gpdF/gpdR (Marcos P. S. Câmara, et al. 2002). The PCR products were sequenced and deposited in GenBank (accession NO.OK560128 and OK627661 ). The similarity of ITS sequences between the isolate and MH843733 (Stemphylium eturmiunum strain ST14) was 100%, and that of gpd sequences between the isolate and MH843728 (Stemphylium eturmiunum strain ST20) was 100%. The maximum parsimony method of Mega7.0 was used and demonstrated that the studied isolate converged to the same branch as Stemphylium eturmiunum. Koch's postulates was applied to identify the pathogenicity of this isolate. A disc of P6-3-1-culture on PDA (5 mm in diameter) was placed on apple leaves and fruit wounds. Sterile PDA was used as a control. All plants were kept in a growth chamber at 25-30 ℃. Four days after inoculation, the disease spot was observed on the inoculated sites and fruit, and with the extension of incubation time, the diseased spots continue to grow, and the leaf spots were not limited by the veins. The pathogen was re-isolated from the inoculated leaves and fruit, satisfying Koch's postulates. This pathogen can also cause postharvest rot of sweet cherry (Alice Spadoni, et al, 2020), postharvest rot on tomato (Prencipe Simona, et al, 2021), etc. This is the first report that Stemphylium eturmiunum can cause apple leaf spot and fruit black spot in Yunnan province, China. The apple black spot caused by Stemphylium eturmiunum was accurately identified. By distinguishing between the two similar diseases mentioned above, resistance to the host and management practices can be accrued based on the characteristics of the pathogen, its epidemiological pattern and the choice of an effective chemical fungicide.

Postharvest Rot of Pomegranate Fruit in Southern Italy: Characterization of the Main Pathogens.

Pomegranate (Punica granatum L.) is an emerging crop in Italy and particularly in southern regions, such as Apulia, Basilicata, and Sicily, due to favorable climatic conditions. The crop is affected by several pathogenic fungi, primarily in the field, but also during postharvest phases. The most important postharvest fungal diseases in pomegranate are gray and blue molds, black heart and black spot, anthracnose, dry rot, and various soft rots. The limited number of fungicides allowed for treatment in the field and the lack of postharvest fungicides make it difficult to control latent, quiescent, and incipient fungal infections. Symptomatic pomegranates from southern Italy were sampled and isolated fungi were morphologically and molecularly characterized. The data obtained revealed that various species of Penicillium sensu lato (including Talaromyces genus), Alternaria spp., Coniella granati, and Botrytis cinerea were the principal etiological agents of postharvest pomegranate fruit diseases; other relevant pathogens, although less represented, were ascribable to Aspergillus sect. nigri, Colletotrichum acutatum sensu stricto, and Cytospora punicae. About two thirds of the isolated pathogens were responsible for latent infections. The results obtained may be useful in planning phytosanitary control strategies from the field to storage, so as to reduce yield losses.

First report of Curvularia alcornii as a plant pathogen causing post‐harvest rot of tomatoes

In March 2021 an outbreak of post-harvest rot of tomatoes was observed in fruit collected from the research field of Bidhan Chandra Krishi Viswavidyalaya university campus, West Bengal, India. Approximately 5% of the fruits, cv. Rupali, showed symptoms. Initially, 0.2-0.5 cm round, water-soaked, light brown lesions were observed which gradually increased in radius up to 1-1.5 cm. From the lesions a dark brown to black fungal mass erupted extending up to 2-3mm from the surface of the fruit.

Antifungal properties of hybrid films containing the essential oil of Schinus molle: Protective effect against postharvest rot of tomato

The control of postharvest diseases in tomatoes using hybrid films represents an efficient and inexpensive alternative to avoid economic losses. This work reports on the design, characterization, and in situ application of hybrid films containing chitosan combined with the leaf essential oil of Schinus molle (SmEO) as antifungal agents. These materials were tested in situ on Lycopersicon esculentum cv. uva showing symptoms of soft rot caused by Fusarium oxysporum . According to our analytical conditions, SmEO contained β-phellandrene (15.7%), α-phellandrene (12.1%), elemol (9.1%), apiole (6.4%) and camphene (6.2%) as the most abundant volatiles. Four distinct hybrid films were prepared from 1% chitosan combined with different amounts of SmEO (0.05, 0.1, 0.3 and 0.7% w/v ) to generate four hybrid films named FSm1, FSm2, FSm3 and FSm4. The spectroscopic properties (FT-IR), texture and thickness (SEM), and optical properties (transmittance) of these films revealed that FSm3 and FSm4 had the best physicochemical and in situ antifungal properties to avoid conidial germination and mycelial proliferation of F. oxysporum . The fruits treated with films and essential oil showed a statistically significant delay (>50%) in mycelial growth compared with the control groups (p < 0.05) and a substantial decrease in conidial viability over 6 days (<2%). According to our results, FSm3 and FSm4 significantly avoided the in situ growth of F. oxysporum in tomatoes. Interestingly, all of the films significantly improved fruit firmness in comparison with untreated tomatoes (p < 0.05). The properties of the films reported in this work may improve the shelf life of Lycopersicon esculentum cv. uva. • Films based on chitosan and the essential oil from Schinus molle improved tomato quality. • The physicochemical properties of films suggested their potential use a new fruit barrier. • In situ testes revealed that films delayed the mycelial proliferation of F. oxysporum. • Same tests revealed that films avoided the germination of fungal conidia.

Post-harvest Green Pea Pod Rot Caused by Sclerotinia sclerotiorum in Korea

In June 2017, in Gangneung, Gangwon Province, South Korea, green pea pods exhibited post-harvest rot symptoms. The fungus was isolated from infected pea pods and cultured on potato dextrose agar for identification. The morphological characteristics were examined, sequences of the internal transcribed spacer region and the β-tubulin (βtub) gene were analyzed, and the pathogenicity was confirmed according to Koch’s postulates. The morphology, phylogenetic analysis, and pathogenicity tests confirmed that Sclerotinia sclerotiorum was the causal agent. This study reports the first case of post-harvest green pea pod rot caused by S. sclerotiorum in Korea.

Antifungal activity of thymol against the main fungi causing pomegranate fruit rot by suppressing the activity of cell wall degrading enzymes

Postharvest rot of pomegranate fruits due to fungal infections causes the loss of a significant amount of fruit. In this study, the antifungal effects of thymol on the growth of Aspergillus niger (A. niger) and Penicillium commune (P. commune) were investigated. Molecular sequence analysis of internal transcribed spacer (ITS) gene was used to identify fungal species. ITS of rDNA amplified by polymerase chain reaction (PCR), sequenced and after basic local alignment search, it was registered in GenBank database. The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of thymol for both fungi were 250 and 500 μg mL−1, respectively. Scanning electron microscopy (SEM) in A. niger colony showed cell deformation due to destruction of the cell membrane and loss of cell wall strength at 250 μg mL−1 after 168 h. Evaluation of P. commune cell morphology showed that thymol at 250 μg mL−1 caused superficial wrinkles, bifurcation of hyphal apex and no spore production was observed. Thymol suppressed fungal growth by reducing cellulase and pectinase activity. The smallest cellulase and pectinase halo diameter was found at 125 μg mL−1 of thymol. Thymol at 500 μg mL−1 had a similar fungicidal effect compared with thiabendazole (1500 μg mL−1).

First Isolation and Identification of Neopestalotiopsis clavispora Causing Postharvest Rot of Rosa sterilis and Its Control with Methyl Jasmonate and Calcium Chloride

Postharvest rot is a major issue in fruit. However, the cause of postharvest rot on R. sterilis fruit has not been clarified, and there are few studies on the disease control. In this study, the fungus causing postharvest rot is isolated from the symptomatic R. sterilis fruit, and identified by morphological characteristic, pathogenicity test and molecular identification. Moreover, the effects of methyl jasmonate (MeJA) or calcium chloride (CaCl2) alone and their combination on disease resistance to fruit rot were assessed by the determination of defense-related enzyme activity and other indicators. N. clavispora was identified as the main fungus causing the postharvest rot of R. sterilis fruit. The infected fruits were treated with MeJA and CaCl2, and these partially controlled the disease, were additive in effectiveness when used together, increased retention of vitamin C content and fruit firmness, and both enhanced and improved the retention of PAL, POD and PPO activities. The treatment of 500 μL/L MeJA and 3% CaCl2 resulted in the high inhibition of the disease. To our knowledge, this is the first report of N. clavispora causing R. sterilis fruit rot, and the combined treatment is a promising method for controlling postharvest rot on R. sterilis fruit.

Chemical characterization of essential oil constituents of three selected botanicals and their antimicrobial activity against postharvest rot pathogens of tomato (Solanum lycopersicum L.)

This study investigated chemical constituents of essential oils (EOs) of Thaumatococcus daniellii, Hyptis suaveolens and Hildegardia barteri and their bioactivity against three tomato fruit rot pathogens, Aspergillus terreus, Fusarium oxysporum and Geotrichum candidum. The EOs were extracted using hydro distillation, and their chemical composition was determined by gas chromatography and mass spectrometry, while inhibition was determined by Agar diffusion method. Sesquiterpene hydrocarbons were the major constituents of the EOs of T. daniellii (38.7%), H. suaveolens (49.48%) and H. barteri (48.1%). Caryophyllene, guaiol and β-cubebene were the most predominant constituents of T. daniellii, H. suaveolens and H. barteri EOs, respectively. The EOs effectively inhibited radial growth of the pathogens in the in vitro bioassay and their efficacy was significantly higher than mancozeb. This study has shown the prospects of the EOs and sets the pace for further research in the management of postharvest tomato fruit rot under field conditions.

First Report of Mucor irregularis Causing Postharvest Fruit Rot on Garcinia mangostana in China.

Mangosteen (Garcinia mangostana Linn.) is an evergreen fruit tree grown in Southeast Asia. The fruit of Mangosteen is dark purple spherical with a diameter of 5 to 8 centimeter, a hard rind and white flesh. The seeds and pericarps of the fruit have long been utilized as a traditional medicine in Southeast Asia (Ovalle-Magallanes et al. 2017). Surveys were conducted during the months from March to April 2019 of fruit markets located in Wujing Town, Shanghai (31°02'33″ N, 121°28'04″ E), one noteworthy postharvest disease was observed. Overall 116 fruit samples were collected from 5 different supermarkets, 10.3% (12) of the fruits were found infected with the same symptoms during cold storage. The infected fruits were subjected to pathogen isolation and characterization. The junction areas between diseased and healthy tissues were cut into small pieces of diameter 5 mm, which were surface sterilized with 0.5% NaClO solution for 3 min, followed by rinsing in sterile water thrice, and then the tissues were placed on sterilized potato dextrose agar (PDA) plates supplemented with 50 μg/ml streptomycin. The colony edges of resulted fungal cultures were cut and removed to new PDA plates for purification after 2 days incubation at 28°C. After 3 rounds of subculture, a total of 19 purified isolates were obtained and these isolates showed similar phenotypes in morphological characters. The fungal colonies were fast growing (25 mm per day at 28°C on PDA plates) and were circular shaped with massive aerial hyphae, white to ochraceous color. Non-septate hyphae were hyaline and the septa were found only at the branching points. Sporangiophores arose from hyphae or from stolons. Sporangiospores were hyaline, smooth-walled, mostly subspherical to ellipsoidal, and measured 1.34 to 2.73 µm (width) and 3.12 to 6.19 µm (length) (1.98 ± 0.36×4.46 ± 0.69, n=100)(Alvarez et al. 2011). Three independent isolates (GM-07, GM-16, GM-19) were selected used for further genetic analysis and pathogenicity test. The internal transcribed spacer (ITS) and the D1/D2 region of the nuclear large subunit ribosomal RNA gene (LSU) were amplified by primers ITS1/ITS4 and primers NL1/LR3, respectively (Walther et al. 2013). BLASTn analyses of ITS (MN081612, MN081613, MN087659) and LUS (MN080423, MN080422, MN080432) sequences exhibited 99 to 100% identify to Mucor irregularis strains HZ640, extr25 and CBS977.68 (Accession numbers KC461926, MH510278 of ITS and JX976214 of LUS). These observed morphological features and molecular identification indicated that the pathogen possessed identical characteristics as Mucor irregularis Stchigel, Cano, Guarro & Ed. Alvarez (Alvarez et al. 2011). To conduct a pathogenicity test (Al-Sadi et al. 2014), 20 µl of spore suspension (106 spores ml-1) were inoculated into five healthy mangosteen fruits by sterile syringes, meanwhile another five healthy fruits were injected with sterilized water as controls. All inoculated fruits were kept in sealed plastic boxes at 28°C with a 12L/12D illumination cycle and 90% relative humidity. After about 3 to 7 days, the inoculated materials showed similar symptoms to those on the original diseased fruits, while control samples remained symptomless. M. irregularis was successfully re-isolated from symptomatic fruits, fulfilling Koch's postulates. Only three plant species that can be infected by M. irregularis have been reported so far, including key lime from Oman (Al-Sadi et al. 2014), corn from China (Peng et al. 2015), and sorghum from Brazil (Lima et al. 2018). To our knowledge, this is the first report of M. irregularis causing the postharvest rot on mangosteen fruit in China.

Arginine induces the resistance of postharvest jujube fruit against Alternaria rot

Abstract In order to explore the effects of arginine (Arg) treatment on postharvest rot of jujube caused by Alternaria alternata (A. alternata), winter jujube was treated with different concentrations of Arg (0, 20, 200, and 1000 μmol/L). Results showed that Arg treatment substantially inhibited the expansion of lesion diameter, and jujubes treated with 200 μmol/L Arg had the smallest lesion diameter. In vitro experiments demonstrated that Arg could not inhibit spore germination and mycelial growth of A. alternata. Further experimental results showed that Arg treatment reduced the production rate of O2–. and H2O2 content and improved the activities of superoxide dismutase, catalase, peroxidase, and ascorbate peroxidase in comparison with the control; Arg treatment enhanced the activities of chitinase and β-1,3-glucanase. Furthermore, Arg treatment significantly increased the activity of phenylalamine ammonia lyase and the contents of flavonoids, phenolics, and lignin. Results indicated that, although Arg could not directly inhibit the growth of pathogenic fungi as a fungicide, it can induce resistance to Alternaria rot by maintaining the balance of reactive oxygen species, increasing the activities of pathogenesis-related protein, and promoting the phenylpropane metabolism in jujube fruit tissue. Therefore, Arg treatment can be a novel measure for inducing the resistance of jujube to postharvest Alternaria rot.

Volatiles produced by Streptomyces spp. delay rot in apples caused by Colletotrichum acutatum.

Volatile organic compounds (VOCs) produced by microorganisms may prevent postharvest rot in fruits. Here, it was examined if VOCs from different species of Streptomyces can control infection in apples caused by the fungal pathogen Colletotrichum acutatum. Incubation of C. acutatum-infected apples in semi-closed boxes with actively growing strains of three Streptomyces (S. coelicolor, S. diastatochromogenes and Streptomyces strain 2R) showed that VOCs reduced rot areas of the apples by 45-66% after 8 days and 39-57% after 10 days, relative to infected apples incubated without Streptomyces. No differences in inhibition among the three strains were seen. In contrast, a mutant strain of Streptomyces that lacks major genes involved in biosynthesis of secondary metabolites, did not reduce development of rot in the apples. Furthermore, Streptomyces VOCs reduced radial hyphal growth of C. acutatum on agar. Several of the VOCs produced by three Streptomyces strains have previously shown fungicidal properties. Although the specific VOCs being active in inhibition of C. acutatum remain to be determined, VOCs may have a great potential as biofumigants to minimize postharvest diseases in fruits.

Óleo essencial de Eucalyptus staigeriana no controle de podridões fúngicas no período pós-colheita e na análise sensorial de uvas

Abstract The objective of this work was evaluate the effect of Eucalyptus staigeriana essential oil on Colletotrichum gloeosporioides and Greeneria uvicola mycelial growth and conidia germination on grapes, as well as its potential for the control of postharvest rot diseases and its effect on the organoleptic properties of grapes. The essential oil (EO) showed in vitro antifungal activity against both pathogens, with fungicidal effect on mycelial growth and on conidia germination at the concentrations of 1.0 and 0.5 μL mL−1, respectively. The EO volatile compounds had a fungistatic effect on the mycelial growth of C. gloeosporioides and a fungicidal effect on G uvicola. At postharvest, the EO reduced the incidence of ripe rot up to 75% and 86% in the preventive and curative treatments, respectively, and the incidence of bitter rot up to 54% in the curative treatment. Since the EO does not affect significantly grape sensory properties, it does not affect the consumption intention of grapes treated with the EO. The EO of E. staigeriana is efficient in the in vitro control of both pathogens; moreover, it is also efficient in the control of the incidence of postharvest fungal rot diseases, mainly in the curative treatment.

Susceptibility of Rosaceous Pome and Stone Fruits to Postharvest Rot by Paecilomyces niveus.

Paecilomyces rot of apples is a postharvest disease caused by Paecilomyces niveus, a problematic spoiling agent of fruit juices and derivatives. Processing fruits infected with Paecilomyces rot can lead to juices contaminated with P. niveus ascospores. These ascospores are heat resistant and may survive food processing and germinate in finished products. Because the fungus produces the mycotoxin patulin, juice spoilage by P. niveus is an important health hazard. Little is known about the disease biology and control mechanisms of this recently described postharvest disease. The range of fruit products contaminated by P. niveus and patulin led us to hypothesize that the host range of Paecilomyces rot is broader than previously thought. Following Koch's postulates, we determined that multiple untested rosaceous fruits and popular apple cultivars are susceptible to Paecilomyces rot infection and that these infected fruits contain significant levels of patulin. We also observed that two closely related food spoiling fungi, Paecilomyces fulvus and Paecilomyces variotti, were unable to infect, cause symptoms in, or grow in wounded fruits. Therefore, we challenge the assumption that P. niveus spoilage inoculum is introduced to foods solely through environmental sources, and we show that other economically important rosaceous fruits, peaches, pears, sweet cherries, and sour cherries, are susceptible to infection and can also be sources of spoilage inoculum. Our results highlight the unique abilities of P. niveus to infect a variety of fruits, produce patulin, and form resistant spores capable of spoiling normally shelf-stable products.

Nanotechnology-Based Advancements in Postharvest Management of Horticultural Crops

Horticulture is a branch of Agricultural science where it is defined as the science and art of cultivating and handling fruits, vegetables, ornamental plants and several plants having unique medicinal and aromatic values. Horticultural crops provide farmers with high income and have good export quality, but they have a concern about postharvest losses. Hence, increasing productivity and decreasing post-harvest losses by using scientific studies and techniques like biotechnology and nanotechnology could be the simplest possible solution to the above-mentioned problems. Using nanotechnology which is having the characteristics of nanoparticles is proven to be very useful in science and technological applications. Nanotechnology-based formulations increase the product quality and the shelf life of horticultural products and provide multiple ways of inhibiting the growth and development of microorganisms. It is precisely a new edible packaging coverage (film) that controls the exchange of gases and prevents damage from harmful rays such as ultraviolet radiation to a greater extent. Increasing strength by using nano biosensors for labeling products is considered a fundamental process to automated control of storage products. Postharvest rotting of vegetables is recognized to be an oxidative reaction and microbial deterioration as well. This review will address all such nanotechnology-based advancements for minimizing post-harvest losses of horticultural crops and enhancing the socio-economical progress of growers in particular.

Identification and characterization of fungi pathogen causing fruit rot disease of watermelon (Citrullus lanatus)

Postharvest rot caused by Fusarium species is considered one of the most devastating diseases limiting production and commercialization of water melon worldwide. Two economically important fungi species are generally responsible for fruit rot in watermelon. The aim of this study was to identify and characterize the fruit rot causing pathogen in watermelon in Ibadan Nigeria. Symptomatic watermelon leaves and fruits expressing severe wilt and rot were collected and cultured on solidified Potato dextrose agar. Pure culture of the isolated pathogen was identified based on morphological and molecular characterization as a new species of Fusarium viz. Fusarium chlamydosporium. The pathogenicity of F. chlamydosporum confirmed on watermelon and golden melon fruits revealed its capability to cause infection on healthy fruits. Golden melon fruits artificially inoculated with F. chlamydosporum isolate had higher rot diameter (81.7 mm) than watermelon (40.0 mm), disease incidence and severity also was significantly higher in golden melon with 88.3% and 100.0% respectively. To our knowledge this is the first report of F. chlamydosporum as a causal pathogen of watermelon wilt and fruit rot disease in Nigeria.

First report of post-harvest Fusarium rot of mandarin Citrus reticulata cv. 'Kinnow' caused by Fusarium equiseti in Pakistan.

Citrus reticulata cv. 'Kinnow' mandarin is the most popular and widely grown fruit crop in Pakistan. During 2017, a survey was conducted to the local citrus fruit markets of Faisalabad, Pakistan. Citrus fruits (n=50) exhibiting stem end rot and fruit rot were collected with 15% disease incidence. The stem end region showed light to dark brown lesions and white fungal growth was also observed in the severely infected fruit. Infected fruit were excised into 2mm2 segments, surface disinfected with 1% NaClO, rinsed with sterilized water and dried. Later, these tissues were placed on potato dextrose agar (PDA) medium and subsequently incubated at 25 °C. Purified isolates produced white colonies with beige pigmentation. The frequency of fungal isolation was 47%. Microscopic observations revealed that macroconidia (n=50) had 5 to 6 septations, with a prominent dorsiventral curvature, tapered and elongated apical cell, and a foot shape basal cell. The macroconidia were measuring 22 to 45 × 2.9 to 4.3 µm with an average of 31 × 3.6 µm. However, microconidia were not observed. Chlamydospores were globose, intercalary, solitary, or in pairs, appearing in chains (Leslie and Summerell 2006). For molecular identification, DNA was extracted from all isolates. The internal transcribed spacer region (ITS) ITS1/4 (White et al. 1990), translation elongation factor-1 alpha (TEF) EF1/2 (O'Donnell et al. 1998), and RNA polymerase II subunit 1 (RPB1) (O'Donnell et al. 2013) were amplified using PCR and the product was subsequently sequenced. Based on BLAST analysis, the isolate was identified as Fusarium equiseti (FUS-21). The sequences of the representative isolate FUS-21 were deposited in the GenBank with accession numbers (ITS, MH581300), (TEF, MK203749), and (RPB1, MW596599) showing more than 99% similarity with ITS accession GQ505683, TEF accession GQ505594, and 100% to RPB1 accession JX171481. To determine the pathogenicity, 40 healthy surface disinfested citrus fruit were taken. The fruit were inoculated by creating artificial wounds on the surface with a sterilized needle and 10 μL of 105 spores/mL was deposited in the wounds. In case of control fruit were inoculated with 10 μL sterilized distilled water only, and incubated at 25 °C. All fruit inoculated with the putative pathogen, developed symptoms like the original fruit from which they were isolated. The pathogenicity test was repeated twice. Visible white mycelium appeared at the stem end region and the fruits became dried as the infection progressed. However, the control fruit remained asymptomatic. The pathogen was re-isolated from infected fruit and identified based on morphometric and molecular analysis. Previously we have reported F. oxysporum causing citrus fruit rot in Pakistan (Moosa et al. 2020). This is the first report of F. equiseti causing post-harvest rot of citrus fruits in Pakistan. Kinnow is an important fruit crop of Pakistan with huge export value the management of Fusarium rot is quite important to save the loss of fresh produce.

Molecular Characterization of Post Harvest Fungus Associated with Spoilt Broccoli

Broccoli (Brassica oleracea var. italica) is a nutritional vegetable that looks like a small tree. Despite the fact that it is extensively loaded with arrays of vitamins, minerals, fiber and antioxidants, it has been observed that it has a short lifespan of not more than 2-5 days due to post-harvest deterioration. A study was conducted to isolate and identify the common fungal pathogens causing post-harvest deterioration of broccoli crown. Diseased broccoli crowns were collected from Ogunabali Fruit Garden Market in D-Line, Port Harcourt Local Government Area of Rivers State. Fungal isolates were collected and morphologically identified. The DNA of the most common fungal isolate, BC-3B was molecularly characterized using Internal Transcribed Spacer 4 and 5 (ITS-4 and 5) molecular markers. The morphological studies revealed that the BC-3B isolate was an Aspergillus niger. The BC-3B isolate DNA sequence was aligned using Basic Local Alignment Search Tool for Nucleotide (BLASTN) 2.8.0 version of National Center for Biotechnology Information (NCBI) database. The molecular weight of the DNA of the isolates was over 600base pairs. Based on sequence similarity, it was observed that the broccoli isolate BC-3B was 93% identical to Aspergillus niger. From the above results, these findings showed that Aspergillus niger is the causal fungal pathogen of post-harvest rot of broccoli. Phylogenetic tree was constructed to access the relationship between the isolates obtained from this study. This study has provided information on some of the fungal organisms found in broccoli. It is anticipated that this result will provide information for disease control approach for alleviating the post-harvest losses of broccoli caused by Aspergillus niger and provide a foundation for further study of possible harm of consuming diseased broccoli.