Application of Radiation and Genetic Engineering Techniques to Improve Biocontrol Agent Performance: A Short Review

Biological control is a potential nonchemical method to manage plant pathogens by beneficial microorganisms. To improve antagonistic potential of biocontrol agents, mutation by radiations, chemicals, and genetic manipulations has been used. Genetic techniques and ionizing radiation containing direct or indirect emissions play the greatest role for selection of useful microorganisms to enhance the efficiency of biological systems. Indeed, genetic engineering has a main role in increasing antimicrobial metabolites, host colonization ability, and endurance in micro-ecosystem. Genetic improvement can be achieved by protoplast fusion, genetic modification (GM), and chemical (genotoxic agents) and physical mutations. However, ultraviolet light and ionizing radiations can induce modifications in the genome of an organism. Irradiation, particularly gamma rays, is also applied for controlling postharvest diseases. Indeed, irradiation cannot completely eliminate pathogens, but it might result in cell injury and directly damage the chromosomal DNA of a living cell. This technology has been used for many reasons including disinfestation of foods, reducing foodborne pathogens, and extending shelf life many fruits, vegetables, and nuts. In the current review, we discuss advances in the radiation and molecular genetic techniques with the aim to improve antagonistic potential of microorganisms as it is applied to the suppression of plant pathogens

Occurrence of mycotoxin patulin and polyphenol profile of Nordic apple juices in relation to apple cultivation system and pre-processing storage temperature

The aims of this study were to find out if organic apple juice (AJ) contained higher contents of polyphenols or patulin compared to conventional AJ, and if higher storage temperature before processing increases patulin content in juice. AJ was pressed from Estonian, Danish and Norwegian apples. Additionally, three cultivars from Estonian organic and conventional orchards were stored at 3±2 °C and 9±2 °C before processing. Patulin, polyphenol content and antioxidant capacity were determined in pasteurized juices. In 2015, 33% of conventional (n=6) and 46% of organic (n=11) juices contained patulin; two of the organic juices above the legal limit (191 and 64µg l-1). In 2016, none of the AJs contained patulin. Patulin occurrence was more affected by weather conditions two weeks before harvest than by cultivation system and apple storage temperature. Polyphenol content was higher in organic than in conventional juices and was reduced at higher apple storage temperature

Mulla patogeeni Verticillium dahliae Kleb. ja mulla seenekoosluste iseloomustamine maasikapõldudel

A Thesis for applying for the degree of Doctor of Philosophy in AgricultureStrawberry (Fragaria × ananassa) is a high value crop, and it is grown for its berries in many countries. Strawberry industry in Estonia has been fluctuating due to the limited selection of cultivars suitable for Nordic climate that are also susceptible to several diseases. Due to the complexity of soil systems and the inadequacy of conventional techniques for describing microbial community composition, it is difficult to diagnose and forecast all ongoing diseases. Strawberry fields are often subjected to disease complex involving different soil-borne pathogens. One of the most widespread and destructive diseases of strawberry is Verticillum wilt caused by Verticillium dahliae. So, knowing about the presence and amount of V. dahliae in the soil can be a key factor in determining appropriate management strategies. The general goal of the present dissertation was on assessment of V. dahliae inoculum density and soil fungal communities associated with Estonian strawberry fields in order to a better understanding of population dynamics. Additionally, finding new potential biocontrol agents was also important in terms of their implications for biological control. In this respect, PCR-based methods used for detection and/or quantification of the most widespread strawberry pathogens were systematically reviewed (I) and then general description of different real-time PCR (rtPCR) chemistries applied in plant pathology was illustrated (II). Also, a rtPCR assay combined with a conventional technique was developed for detection and quantification of V. dahliae directly from strawberry plants and soils of different major production areas (Vasula, Rohu, Unipiha, Utsu, Kaie-Mare and Marjamaa) in Estonia (III, IV). Moreover, soil fungal communities in same strawberry production areas (except Kaie-Mare) were investigated using Illumina-based sequencing as the first study, which may improve available management strategies against strawberry soil-borne diseases (V). Lastly, the antagonistic potential of a native Ttichoderma harzianum collected from Estonian fields as well as Gliocladium catenulatum isolated from a bifungicide toward V. dahliae was assessed with the aim of protecting biological resources (VI, VII). As the first study in Estonia, the newly developed rtPCR protocol efficiently enabled detecting and quantifying V. dahliae in strawberry plants and soils in which, 10.48 pg μl−1 of pathogen DNA represented at least one Microsclerotia (MS) per gram of soil, showing a high level of quantification in comparison with other studies. In fact, the presence of V. dahliae in strawberry production areas exhibited considerable variation, being high in samples from Vasula and Marjamaa, moderate in Rohu and Utsu, and low in Unipiha. No V. dahliae was detected from Kaie-Mare district. According to Illumina sequencing of strawberry soils from five commercially production sites, a high number of sequence matched V. dahliae in most samples particularly from soils with diseased plants (Vasula and Marjamaa) and so proved the interpretation of earlier estimates using rtPCR. Arbuscular mycorrhizal fungi were more abundant in areas with healthy plants (Rohu, Utsu and Unipiha), which may highlight their suppressive role against fungal pathogens. Moreover, biocontrol ability of T. harzianum isolates and G. catenulatum over V. dahlia was proven, representing as useful candidates for biocontrol of one of the most economically important pathogen of strawberry. The current dissertation provided important insights into the rtPCR as a valuable quantitative technique for diagnosis of important pathogens such as V. dahliae with high accuracy and rapidity as well as presented a comprehensive study to date on soil fungal communities in Estonian strawberry fields, which may help to achieve better understanding of the biological characteristics of soil in development of diseases. The data described within this dissertation may provide useful information for growers and agricultural organizations for applying suitable disease management strategies against plant pathogenic.Aedmaasikas (Fragaria × ananassa) on hinnatud marjakultuur, mida kasvatatakse paljudes riikides. Eestis on maasikakasvatuse maht aastate lõikes varieeruv, kuna kohalikule kliimale sobivate ja haiguskindlate sortide valik on piiratud. Paljud mullapatogeenid on maasikahaiguste põhjustajateks. Kõige levinum ja kõige suurema kahju tekitaja on Verticillium dahlia, mis põhjustab närbumistõbe. Maasikahaiguste määramine on keeruline, kuna mulla mikrobioom on kompleksne ja haigustekitajate määramiseks sobivate meetodite valik on piiratud. Parima agrotehnoloogia leidmiseks on vajalik teada V. dahliae kogust mullas. Uurimistöö eesmärgid: 1) Analüüsida PCR-meetodite täpsust ja usaldusväärsust enamlevinud maasika patogeenide kvalitatiivsel ja kvantitatiivsel määramisel (I, II). Hüpotees: PCR-meetodid sobivad maasika patogeenide detekteerimiseks ja identifitseerimiseks, kuid kvantitatiivne reaalaja PCR (rtPCR) on sobilik meetod patogeenide arvukuse määramiseks. 2) Koostada tundlik ja spetsiifiline rtPCR protokoll, et kvantitatiivselt määrata V. dahliae arvukus maasikatest ja mullast Eestis (III). Hüpotees: rtPCR protokoll võimaldab iseloomustada V. dahliae arvukust maasikates ja mullas. 3) Hinnata V. dahliae mikrosklerootiumite arvukust mullas kahe meetodiga, klassikaline morfoloogiapõhise meetodi ja kvantitatiivse rtPCR-iga ja võrrelda saadud tulemusi ((IV). Hüpotees: rtPCR võimaldab kiiremat ja tundlikumat kvantifitseerimist, määrates patogeenid ka terve välimusega taimedes. 4) Iseloomustada Eesti maasikapõldude muldade seenekooslusi järgmise põlvkonna sekveneerimise abil(V). Hüpotees: Järgmise põlvkonna sekveneerimine võimaldab laiaulatuslikke seenekoosluste avastamist. 5) Hinnata G. catenulatum ja T. harzianum isolaatide võimekust kontrollida V. dahliae levikut. Hüpotees: G. catenulatum ja T. harzianum on efektiivsed V: dahliae tõrjel. Metoodika Maasika patogeenide määramiseks kasutatud ja avaldatud PCR protokollidest tehti süstemaatiline ülevaade (I). Süstemaatilise otsingustrateegiaga leiti 22 otsingukriteeriumitele vastavat artiklit. Taime ja mullaproovid koguti aastatel 2014-2015 aedmaasika põldudelt, mis asusid vastavalt Vasula, Rõhu, Unipiha, Utsu, Kaie-Mare ja Marjamaa piirkonnas. Kasvatav maasikasort oli Sonata. Seenpatogeenid ja V. dahliae arvukus määrati juure ristlõikest ja ühest grammist mullast morfoloogiliste tunnuste alusel (III, IV, V, VI). G. catenulatum eraldati biopreparaadist Prestop® (VII). Kõikide V. dahliae isolaatide patogeensus testiti kahe meetodiga: juurte ja mulla inokulatsiooniga (VI). DNA eraldati puhaskultuuridest, taimedest ja mullast ning selle kvaliteeti kontrolliti cPCR abil (ITS1/ITS4 praimeritega). V. dahliae kvantifitseerimiseks disainiti vastavad praimerid (VD-rtPCR-F/VD-rtPCR-R) (III, IV). Reaalaja PCR protokolliga (CYBRGreen reaktsioonikeemiaga) kvantifitseeriti V. dahliae. Patogeeni DNA kontsentratsiooni määramise standardkõvera amplifitseerimise efektiivsus oli 95.67% ja kõige madalam amplifitseeritud DNA kontsentratsioon 0.93 pg μl−1 (III, IV). Uue põlvkonna sekveneerimise meetodit rakendati seenekoosluste iseloomustamiseks, amplifitseerides ja järjestades ITS1 regiooni seene rRNA geenis (V). Mullast eraldatud T. harzianum ja biofungitsiidist eraldatud G. catenulatum isolaatide patogeeni allasurumisevõimet testiti in vitro ja in vivo (VI, VII). Statistilised analüüsid teostati R programmiga ja varieeruvusanalüüsid (ANOVA) programmiga MSTATC (v. 1.42). Tulemused ja arutelu Süstemaatiliselt koguti ja analüüsiti kõik maasika patogeenide määramiseks seni kasutatud PCR-põhised meetodid, täpsemalt iseloomustati rtPCR protokolle (I, II). Kogutud info alusel töötati välja rtPCR protokoll, millega määrati kvalitatiivselt ja kvantitatiivselt Eesti suuremate maasikatootjate põldudelt (Vasula, Rõhu, Unipiha, Utsu, Kaie-Mare and Marjamaa) kogutud mulla ja taime proovidest V. dahliae sisaldus (III, IV). Kasutatud ITS regiooni spetsiifilised praimerid on laialt kasutusel patogeenide määramiseks (Lees et al. 2002; Luchi et al. 2005). Tõestust leidis ka ITS praimerite kõrge spetsiifilisus V. dahliae määramisel väga heterogeensest proovist. rtPCR võimaldas määrata isegi terve välimusega taimedes 11.05 pg μl−1 patogeeni DNA-d. See näitab, et paljud taimed võivad olla haigustekitaja varjatud kandjad (Markakis et al. 2009). Kasutades soil plating meetodit, leiti et V. dahlie arvukus varieerus ühes grammis mullas 1 kuni 13 mikrosklerootiani. Teiste meetoditega võrreldes oli rtPCR meetod väga kõrge tundlikkusega, sest 10.48 pg μl−1 patogeeni DNA-d leiti proovidest, mis klassikalise meetodiga ühtegi tulemust ei andnud. V. dahliae kontsentratsioon maasika tootmispõldudel oli väga varieeruv. Vasula ja Marjamaa proovides oli haigustekitaja sisaldus väga kõrge, Rõhu ja Utsu proovides oli see keskmine ja Unipiha proovides madal. Kaie-Mare piirkonnast ei tuvastatud ühtegi V. dahlia patogeeni. Lisaks iseloomustati mulla seenekooslusi uue põlvkonna sekveneerimise abil (V). Identifitseeriti palju taksonoomiliselt erinvaid seenerühmi. Kõige rohkem esines rühmi Ascomycota ja Basidiomycota (V), mis on ühtlasi ka üle maailma kõige enamesindatud seened mullas (Gomes et al., 2003). Tulemused kinnitasid rtPCR protokolliga saadud tulemusi ja kõige rohkem V. dahlia järjestusi leiti muldadest, kus oli palju haigustunnustega taimi (Vasula ja Marjamaa). Lisaks leiti teisi maasikapatogeene: F. solani, R. solani ja C. truncatum. Arbuskulaarsete mükoriisaseente (Rhizophagus irregularis ja Glomus hoi) sisaldus oli kõrgem seal, kus domineerisid terved taimed (Rõhu, Utsu ja Unipiha), mis ilmselt viitab mükoriisaseente võimele haigustekitajaid alla suruda (Nallanchakravarthula et al., 2014). Samas on leitud, et vastupidi, arbuskulaarsete mükoriisaseente arvukus ei pruugi alati taimede haigustele vastuvõtlikkust mõjutada (Xu et al., 2012b). Edasine uurimistöö peab analüüsima keskkonna rolli taime haigestumisel. Analüüsiti Eesti põllumullast eraldatud Trichoderma harzianum ja biofungitsiidist eraldatud Gliocladium catenulatum isolaatide potentsiaalset negatiivset mõju V. dahliae kasvule (VI, VII). Leiti, et T. harzianum ja G. catenulatum olid võimelised V. dahlia kasvu pärssima in vitro ja in vivo, mis teeb neist head kandidaadid maasika patogeenide biotõrjeks. Leiti, et mõlemad isolaadid sünteesisid erinevaid lenduvaid ja mittelenduvaid metaboliite, mille negatiivne mõju takistas V. dahliae mütseelkasvu (mycelial growth). Varasemalt on samuti leitud, et T. harzianum toodab erinevaid antibiootilise toimega metaboliite, mis inhibeerivad taime patogeenide kasvu (Siddiquee et al., 2012). Järeldused • PCR protokollid võimaldavad maasika patogeene tuvastada; neist rtPCR protokoll on kõige lootustandvam meetod, mis võimaldab enamlevinud maasika patogeene määrata kvalitatiivselt ja kvantitatiivselt (I, II). • Välja töötatud rtPCR protokoll võimaldas efektiivselt ja täpselt määrata ja kvantifitseerida V. dahliae sisaldust nii maasika taimedes (nii haigussümptomite kui ka ilma sümptomita taimedes) kui ka mullas (III, IV). • V. dahliae DNA kontsentratsioon 10.48 pg μl−1 vastab ühele mikrosklerootiale grammis mullas (IV). • Uue põlvkonna sekveneerimise rakendamine võimaldas iseloomustada maasikapõldude mulla seenekooslusi (V). Mida rohkem oli haigustunnustega taimi, seda rohkem esines mullas V. dahliae järjestusi. • Arbuskulaarsete mükoriisaseente sisaldus oli kõige kõrgem neis mullaproovides, mis oli kogutud põldudelt, kus olid valdavalt terved taimed (Rõhu, Utsu ja Unipiha), viidates nende võimalikule rollile haigustekitajate tagasitõrjumisel (V). • Mullast eraldatud T. harzianum ja biofungitsiidist eraldatud G. catenulaum olid võimelised takistama V. dahliae kasvu nii in vitro, kui in vivo, mis tähendab, et neil on potentsiaali biotõrjes. Käesoleva doktoritöö tulemused näitasid V. dahlia näitel, et rtPCR meetod on tundlik ja täpne maasika patogeenide kvantitatiivseks määramiseks. Töös iseloomustati ka Eesti maasikapõldude seenekooslusi, mis on tähtis, et mõista paremini mulla bioloogia rolli haiguste tekkimisel ja arenemisel. T. harzianum ja G. catenulaum osutusid headeks biofungitsiidideks, mis võimaldasid V. dahliae kasvu pidurdada. Saadud tulemused aitavad maasikakasvatajal paremini määrata põllul haigustekitajaid, mis aitavad kiiremini ja täpsemini valida sobivaid võtteid patogeenide kontrolli all hoidmiseks.Publication of this dissertation is supported by the Estonian University of Life Sciences, Ministry of Education and Research and the Doctoral School of Earth Sciences and Ecology

Combination of Potassium Phosphite and Reduced Doses of Fungicides Encourages Protection against <i>Phytophthora infestans</i> in Potatoes

Late blight caused by the oomycete Phytophthora infestans is considered the biggest threat to potato farming worldwide. For susceptible cultivars, the disease is often managed by frequent applications of fungicides to reduce yield loss. The use of bio-based compounds that interfere with biologically active systems is an innovative strategy for improving disease management. In the present work, the control of P. infestans infection on potatoes by potassium phosphite (KPhi) combined with recommended and reduced doses of active ingredients (Ais) from different fungicides was evaluated. The protective effects of different combinations were initially assessed in vivo and subsequently compared with a greenhouse screening. The active ingredients cyazofamid (CFD) and mancozeb (MCB), used at recommended and reduced doses, were less effective at reducing P. infestans infections than when combined with KPhi. In greenhouse trials, CFD, mandipropamid (MPD) and MCB at recommended doses were the most effective treatments when combined with KPhi; meanwhile, the combination of KPhi with azoxystrobin (AZ), benthiavalicarb-isopropyl/mancozeb (ISO/MCB), and CFD at reduced doses exhibited strong protective activity compared to other similar combinations. This decreased the severity of infection by P. infestans up to ~89%. Greenhouse experiments also demonstrated that a combination of KPhi and CFD at both doses caused the highest reduction in disease severity (up to ~90%) within 35 days of infection. In microplot experiments, KPhi delayed the progression of late blight in susceptible potato varieties; therefore, in the combined treatments AUDPC values were significantly lower than those obtained after applications with CFD doses, providing sufficient protection against late blight. Our data suggest that optimizing the formulation with addition of KPhi could result in a lower recommended dose. This would result in a reduction of the active compounds of the fungicides in potato farming. Furthermore, the impact of KPhi on late blight development makes it a potential component for incorporation into an integrated pest management system

Combination of Potassium Phosphite and Reduced Doses of Fungicides Encourages Protection against Phytophthora infestans in Potatoes

Late blight caused by the oomycete Phytophthora infestans is considered the biggest threat to potato farming worldwide. For susceptible cultivars, the disease is often managed by frequent applications of fungicides to reduce yield loss. The use of bio-based compounds that interfere with biologically active systems is an innovative strategy for improving disease management. In the present work, the control of P. infestans infection on potatoes by potassium phosphite (KPhi) combined with recommended and reduced doses of active ingredients (Ais) from different fungicides was evaluated. The protective effects of different combinations were initially assessed in vivo and subsequently compared with a greenhouse screening. The active ingredients cyazofamid (CFD) and mancozeb (MCB), used at recommended and reduced doses, were less effective at reducing P. infestans infections than when combined with KPhi. In greenhouse trials, CFD, mandipropamid (MPD) and MCB at recommended doses were the most effective treatments when combined with KPhi; meanwhile, the combination of KPhi with azoxystrobin (AZ), benthiavalicarb-isopropyl/mancozeb (ISO/MCB), and CFD at reduced doses exhibited strong protective activity compared to other similar combinations. This decreased the severity of infection by P. infestans up to ~89%. Greenhouse experiments also demonstrated that a combination of KPhi and CFD at both doses caused the highest reduction in disease severity (up to ~90%) within 35 days of infection. In microplot experiments, KPhi delayed the progression of late blight in susceptible potato varieties; therefore, in the combined treatments AUDPC values were significantly lower than those obtained after applications with CFD doses, providing sufficient protection against late blight. Our data suggest that optimizing the formulation with addition of KPhi could result in a lower recommended dose. This would result in a reduction of the active compounds of the fungicides in potato farming. Furthermore, the impact of KPhi on late blight development makes it a potential component for incorporation into an integrated pest management system

Biocidal activity of plant-derived compounds against Phytophthora infestans : an alternative approach to late blight management

The current study aimed to evaluate the potential biocontrol capabilities of different essential oils (EOs) including juniper, tea tree, clove, thyme, cinnamon, turmeric, pepper and rosemary at application rates of 0.41, 0.83, 1.66, 3.33 and 6.66 mu L mL(-1 )for control of Phytophthora infestans; the causal agent of potato late blight in vivo and under greenhouse conditions. In particular we tested the highly pathogenic genotype, EU-13-A2. Plant extract Timbor (R) (Thymus vu/garis-derived compound) was similarly applied as an alternative reference to the essential oils. The effectiveness of plant compounds was initially determined by measuring the effective dose (ED50) reducing P. infestans sporangial germination by 50%. Among the nine tested compounds, thyme and tea tree EOs showed the strongest inhibitory effect on sporangial germination, with ED50 values between 0.12 and 0.37 mu L mL(-1), compared to the fungicide control Ranman-top (R) (160 g L-1 Cyazofamide). Clove EO, thyme EO and Timbor (R) significantly inhibited P. infestans mycelial growth even at the lowest concentration tested (0.41 mu L-1). Inhibitory effects of compounds were also assessed in response to late blight in vivo, and the results were then compared with greenhouse screening. In the detached-leaflet assay, pepper, rosemary EOs and Timbor (R) showed reduction of disease development at all concentrations except for 0.41 mu L mL(-1). In dose-responses, rosemary, thyme EO and Timbor (R) were the most effective compounds in reducing late blight (more than 80% disease reduction) over time. Within a 35-day period, these compounds also resulted in the lowest level of disease severity in susceptible potato plants (cv. Bintje) when treated 12 h before inoculation, demonstrating their protective effect against late blight under greenhouse conditions. This study presents evidence for the potential use of commercial plant derived compounds as botanical fungicides against P. infestans to control infection on potato plants

Green leaf volatile confers management of late blight disease : a green vaccination in potato

Yield losses of crops due to plant pathogens are a major threat in all agricultural systems. In view of environmental issues and legislative limitations for chemical crop protection products, the need to design new environmentally friendly disease management strategies has gained interest. Despite the unique capability of green leaf volatiles (GLVs) to suppress a broad spectrum of plant pathogens, their capacity to control the potato late-blight-causing agent Phytophthora infestans has not been well studied. This study addresses the potential role of the GLV Z-3-hexenyl acetate (Z-3-HAC) in decreasing the severity of late blight and the underlying gene-based evidence leading to this effect. Nine-week-old potato plants (Solanum tuberosum L.) were exposed to Z-3-HAC before they were inoculated with P. infestans genotypes at different time points. These pre-exposed potato plants exhibited slower disease development after infection with the highly pathogenic genotype of P. infestans (EU-13-A2) over time. Qualitative assessment showed that the exposed, infected plants possessed significantly lower sporulation intensity and disease severity compared to the control plants. Hypersensitive response (HR)-like symptoms were observed on the treated leaves when inoculated with different pathogen genotypes. No HR-like lesions were detected on the untreated leaves after infection. It was shown that the transcript levels of several defense-related genes, especially those that are involved in reactive oxygen species (ROS) production pathways were significantly expressed in plants at 48 and 72 h postexposure to the Z-3-HAC. The current work provides evidence on the role of Z-3-HAC in the increased protection of potato plants against late blight through plant immunity and offers new opportunities for the sustainable control of potato diseases