The Use of the Polish Germplasm Collection of Nicotiana tabacum in Research and Tobacco Breeding for Disease Resistance

The Polish germplasm collection of Nicotiana tabacum was started in the 1920s. Up to now, more than eight hundred accessions originating from different regions of the world have been gathered in the collection. It includes valuable breeding lines and obsolete cultivars, among them cytoplasmic male-sterile lines. Numerous cultivars are rich sources of features desired in tobacco breeding. Therefore, the accessions are continually characterised in terms of their various features, one of the most important of which is disease resistance. Much research is being done to explain the nature of resistance and its genetic basis. Moreover, cultivars with good agronomic characteristics are used in wide hybridisation, being recipients of resistance genes from wild species or are genetically modified with transgenes conditioning resistance. The biological diversity of cultivars also allows a proper selection of plant material for pathogen studies, while the large number of the accessions facilitates research into the conditions for long seed storage. Numerous examples of the use of Polish tobacco germplasm in research and breeding, specifically in disease resistance, have been presented in this paper

Diversification of resistance response of selected tobacco cultivars (Nicotiana tabacum) depending on the used potato virus Y isolates

Wirus Y ziemniaka (Potato virus Y, PVY) powoduje brunatną nekrozę nerwów liści tytoniu. W obrębie gatunku Nicotiana tabacum odporność na PVY warunkowana jest pojedynczym recesywnym genem va powstałym w wyniku delecji w genie podatności Va. Odporność tę posiada wiele odmian uprawnych tytoniu, jednak może być ona przełamywana przez zjadliwe izolaty PVY, dlatego konieczne jest poszukiwanie nowych źródeł odporności lub łączenie czynników odporności już poznanych. Celem pracy była identyfikacja odmian mających odporność typu va oraz ocena jej skuteczności wobec czterech izolatów PVY, a także poszukiwanie nowych czynników odporności. Przebadano odporność 25 odmian tytoniu poprzez wykonanie testów biologicznych metodą inokulacji izolatami PVY w warunkach szklarniowych oraz zastosowanie testu DAS-ELISA w celu potwierdzenia obecności wirusa w inokulowanych roślinach. Identyfikację genu va prowadzono metodą PCR poprzez amplifikację dwóch markerów genu podatności Va. W odmianach posiadających gen odporności va nie dochodziło do amplifikacji produktów. Najwyższą odpornością charakteryzowała się odmiana VAM, która nie została porażona słabszymi izolatami, chociaż izolaty silne spowodowały nekrozę nerwów. Niższą odpornością cechowało się 6 odmian, które nie wykazały objawów chorobowych po zastosowaniu izolatu słabego. Pozostałe odmiany posiadające czynnik typu va zostały porażone przez wszystkie izolaty. Kolejnych pięć odmian, posiadających gen podatności Va, wykazało objawy tolerancji na zakażenie użytymi izolatami PVY. Odmiana Węgierski Ogrodowy, pomimo obecności genu podatności (Va), nie została porażona przez izolat słaby.Potato virus Y (PVY) causes tobacco veinal necrosis. It be-longs to the group of ten most dangerous plant viruses respon-sible for large economic losses. Within the species Nicotiana tabacum, the resistance to this virus is conditioned by the single recessive va gene resulting from a deletion in the susceptibility Va gene. Many tobacco cultivars have this resistance, including VAM, Wiślica and Virginia SCR (V. SCR). However, their resist-ance is overcome by the virulent PVY isolates, so it is necessary to search for new sources of resistance or combine ones already known. The aim of the study was to identify cultivars with va resist-ance, and to assess its effectiveness depending on the used PVY isolate. Research also included the search for new resistance fac-tors within Nicotiana tabacum cultivars. Twenty five tobacco cultivars were tested for resistance to PVY in biological tests by artificial inoculation under greenhouse conditions. Four virus iso-lates with different levels of virulence were used for the studies. After four weeks, disease symptoms were observed, and DAS-ELISA tests were performed using specific antibodies to confirm the presence of the virus in plants. The tested cultivars showed a different degree of PVY susceptibility depending on the used isolate. The identification of the va gene was carried out by using PCR for amplification of two markers of the length 146 and 402 bp. The presence of amplicons indicated the presence of Va sus-ceptibility gene, while cultivars with the va resistance gene did not amplify the products. The highest resistance was character-istic of cv. VAM which was not infected with isolates IUNG 23 and IUNG 17, defined as weak and medium, although the remain-ing isolates, described as strong (IUNG 22 and IUNG20), caused vein necrosis and the presence of the virus was confirmed in plant tissues by a positive DAS-ELISA test result. Six cultivars that did not show disease symptoms only after applying the weak isolate, had a slightly lower resistance. Other cultivars with va resistance were infected by all used PVY isolates. Another five cultivars, after infection with four isolates, showed symptoms of tolerance, i.e. vein clearing and chlorotic spots of a leaf blade, but they did not have vein necrosis, in spite of that molecular tests confirmed the presence of Va susceptibility gene. Moreover, interesting re-sults were noted for cv. Węgierski Ogrodowy, that, despite the presence of the susceptibility gene, was not infected by weak isolate IUNG23. The last group included susceptible cultivars that reacted with vein necrosis to all used PVY isolates. Since virulent PVY isolates are able to break va resistance, the knowledge of the nature and stability of the resistance of cul-tivars is particularly important, especially within Polish cultivars. In addition, the rising number of necrotic isolates in Poland and in the world, capable of breaking existing sources of resistance, can cause an increased use of tolerant cultivars in tobacco breeding

Resistance Response of the Recently Discovered Species Nicotiana mutabilis to Potato virus Y (PVY) and Tomato spotted wilt virus (TSWV) Compared to Other Sources of Resistance

Nicotiana mutabilis is a recently discovered species within the genus Nicotiana. The aim of the present study was to evaluate its resistance to Potato virus Y (PVY) and Tomato spotted wilt virus (TSWV). Molecular analysis was performed to detect the Va gene determining susceptibility to PVY and the SCAR marker associated with resistance to TSWV. Resistance tests were carried out under greenhouse conditions through artificial inoculation with one TSWV and two PVY isolates. In order to confirm the presence of the viruses in plants, DAS-ELISA tests were performed using antibodies against PVY and TSWV. The results indicated the absence of the PVY susceptibility gene and the presence of the TSWV resistance gene in the genome of N. mutabilis. This species was considered tolerant to the two PVY isolates tested because, despite the positive DAS-ELISA results, the infected plants showed vein clearing and chlorotic spots but no vein necrosis. As a result of TSWV inoculation, N. mutabilis showed a hypersensitive response; however, after four months, 30% of the inoculated plants showed systemic infection. This species extends the genetic variation in the genus Nicotiana and, because of its tolerance to PVY and partial resistance to TSWV, it may be a potential source of resistance to these viruses

Diversification of defense response to potato virus Y among alloplasmic tobacco forms

Cytoplazmatyczna męska sterylność (cms) jest wykorzystywana w hodowli roślin w celu otrzymania form niezdolnych do samozapylenia, które wykorzystuje się głównie w hodowli odmian mieszańcowych F1. Cytoplazmatycznie męskosterylne rośliny otrzymuje się w wyniku mutacji w genomie cytoplazmatycznym przez podstawienie w miejsce natywnej cytoplazmy innej cytoplazmy pochodzącej z odmiennego gatunku lub krzyżowanie w obrębie gatunku. U podstaw cms leży niezgodność pomiędzy genomem jądra komórkowego i cytoplazmą. Alloplazmatyczne formy wykształcają zmodyfikowane, niefunkcjonalne męskie organy generatywne lub nie wykształcają ich wcale. Przy tym zmiany morfologiczne mogą obejmować także inne części kwiatu, a niekiedy całą roślinę. Obecność obcej cytoplazmy może wpływać też na inne cechy, w tym na odporność roślin na patogeny. Aby określić wpływ obcej cytoplazmy na odporność tytoniu na wirus Y ziemniaka (PVY), wykonano sztuczne inokulacje tytoniu dwoma izolatami PVY o różnej wirulencji. Testom poddano 13 izogenicznych form cytoplazmatycznie męskosterylnych odmiany Zamojska 4 z cytoplazmą pochodzącą od dzikich gatunków z rodzaju Nicotiana, a także formę z cytoplazmą cms mutanta otrzymanego w obrębie gatunku N. tabacum. Odporność roślin oceniano na podstawie objawów chorobowych oraz wyników testu immunoenzymatycznego DAS-ELISA. Zaobserwowano różnice zarówno w nasileniu objawów chorobowych, jak i w terminie ich wystąpienia od momentu zakażenia roślin. Zróżnicowanie badanych form alloplazmatycznych pod względem odpowiedzi na infekcję wirusową pozwala przypuszczać, że mechanizmy odporności na PVY mogą być związane z określoną formą cytoplazmy.Cytoplasmic male sterility is used in plant breeding to obtainforms incapable of self-pollination, which are utilized mainlyin breeding of hybrid cultivars. It is based on incompatibilitybetween the cell nucleus and cytoplasm because cytoplasmicmale sterile plants are obtained by mutation in the cytoplasmicgenome or by substitution of the native cytoplasm with anotherthat of a different species. Alloplasmic forms develop modified,non-functional male generative organs or do not develop themat all. Morphological changes may involve other parts of flowersand sometimes a whole plant. An alien cytoplasm may alsoaffect other traits, including resistance to pathogens. In order todetermine the effect of alien cytoplasm on tobacco resistance toPotato virus Y (PVY), artificial inoculations with two virus isolatesdiffering in virulence were performed. The test included13 alloplasmic forms of cultivar Zamojska 4 with cytoplasmfrom Nicotiana wild species and a form with cytoplasm fromthe mutant obtained within Nicotiana tabacum. Plant resistancewas assessed on the basis of disease symptoms and DAS-ELISAimmunoenzymatic test results. Differences were observed inthe severity and earliness of disease symptoms. Fertile cultivarZamojska 4 is considered as tolerant to PVY, that is it reacts toinfection with mild symptoms like vein clearing and chloroticspots of the leaves, but not with vein necrosis. In the case ofa combination of cell nucleus from cv. Zamojska 4 and cytoplasmfrom species N. goodspeedii, N. megalosiphon and N.undulata, alloplasmic forms were not completely resistant, butthe level of their tolerance to strong isolate IUNG20 increasedcompared to that in their male fertile isogenomic counterpartand observed symptoms were delayed. In contrast, tolerance ofcms forms with cytoplasm from the species N. eastii, N. occidentalisand N. suaveolens was overcome leading to developingsevere disease symptoms. Unlike the other cms forms, theform with the cytoplasm of N. tabacum mutant reacted witha development of necrotic symptoms to inoculation with isolateIUNG20, but with delayed mild symptoms when inoculatedwith the weaker isolate IUNG21. The diversified reaction ofdifferent isogenomic alloplasmic forms to viral infection suggeststhat the mechanisms of defense responses depended onthe type of cytoplasm

Inheritance of Potato virus Y tolerance introgressed from Nicotiana africana to cultivated tobacco

Potato virus Y (PVY) is an important pathogen of to- bacco. Growing resistant cultivars is the best way to prevent si- gnificant losses of the crop caused by this virus. However, the protection given by the commonly used resistance factor, called va gene, can be overcome by the highly virulent PVY strains. Therefore, tobacco breeding for resistance will benefit from intro- ducing additional PVY resistance/tolerance factors. BPA is a to- bacco breeding line with introgressed PVY tolerance from a wild species N. africana. This trait is effective against a wide range of PVY isolates, including the ones that overcome va resistance. Here, we describe the inheritance of PVY tolerance of BPA. We obtained F1 and F2 plants from reciprocal crosses between BPA and a susceptible tobacco cultivar BP-210. Then we performed me- chanical inoculation tests using sap from PVY infected leaves on both generations of plants. Four weeks later we recorded disease symptoms and subjected all experimental plants to DAS-ELISA tests. All F1 plants developed vein necrosis which confirmed their susceptibility to the virus. The proportion of susceptible and tole- rant plants in the F2 fitted 3:1 ratio which was expected under the assumption that the tolerance is determined by a single, recessive gene. Moreover, the proportion of the susceptible and tolerant in- dividuals did not differ between two F2 populations derived from crosses where BPA was used as a maternal plant or a pollen do- nor, hence cytoplasmic factors do not influence the tolerance of that breeding line

The Occurrence of Fungal Diseases in Maize in Organic Farming Versus an Integrated Management System

Organic farming is becoming increasingly popular because it leads to healthier products. Due to limitations on the use of chemical protection, however, plants may be more susceptible to pathogen attacks. Therefore, the aim of the study was to determine the occurrence of fungal diseases in maize grown in organic versus integrated systems. The field experiment was conducted during the years 2017–2019 in Puławy, Poland. Three maize varieties, Ambrosini, Smolitop and Ricardinio, were cultivated in two fields with a different crop production system. The incidence of fungal diseases, such as northern corn leaf blight, eyespot, common corn rust, corn smut and Fusarium ear rot, was assessed. Fungal isolates were collected from leaves and cobs with disease symptoms and identified microscopically and molecularly. In both cultivation systems, northern corn leaf blight and eyespot were the most common, while corn rust and fusariosis were seen more often in organic cultivation. Alternaria alternata, Fusarium oxysporum, Fusarium poae, Fusarium graminearum and Fusarium sporotrichioides were the fungal species most frequently detected in the two systems. Additionally, Fusarium verticillioides was common in the organic system. Weather conditions, especially heavy rainfall and high air humidity, greatly influenced the incidence of such diseases

Urinary Bisphenol A Concentrations and Parameters of Ovarian Reserve among Women from a Fertility Clinic

Background: Human exposure to environmentally widespread endocrine disruptors, especially bisphenol A (BPA), has been suggested to affect reproductive health. Animal studies indicate that BPA may play a role in the process of reproduction and impact on maturing oocytes, meiotic cell division or fertilization rate. Nevertheless, data regarding the effects of exposure to BPA on women’s ovarian function are still limited. Therefore, the aim of the current study is to assess the effects of environmental exposure to BPA on ovarian reserve. Methods: The study participants consisted of 511 women in reproductive age (25–39 years) who attended an infertility clinic for diagnosis, due to the couples’ infertility. BPA urinary concentrations were assessed by the validated gas chromatography ion-trap mass spectrometry method. The ovarian reserve was assessed using ovarian reserve parameters: Hormones concentrations: E2 (estradiol), FSH (follicle stimulating hormone), AMH (anti-Müllerian hormone), and AFC (antral follicle count). Results: In the present study, the negative association between BPA urinary concentrations and AMH (p = 0.02) and AFC (p = 0.03) levels was found. Exposure to BPA was not related to other examined parameters of ovarian reserve (FSH, E2). Conclusions: Our results suggest that BPA exposure may affect women ovarian reserve parameters and reduce ovarian reserve. As this is one of the first studies of its kind, the findings need confirmation in a further investigation