Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 77(1): 13-19, 2024 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-2430 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Singh, V. (2024). An account of chromosomal damage in PMCs of stripe rust infected barley. Caryolo- gia 77(1): 13-19. doi: 10.36253/caryolo- gia-2430 Received: December 31, 2023 Accepted: May 26, 2024 Published: July 8, 2024 Copyright: © 2024 Singh, V. This is an open access, peer-reviewed article published by Firenze University Press (ht tps://www.fupress.com/caryolo- gia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. ORCID VS: 0000-0002-4510-7974 An account of chromosomal damage in PMCs of stripe rust infected barley Vivek Singh Department of Botany, Shri Jai Narain PG College, Lucknow, UP-226001, India Email: viveksingh_2@yahoo.com Abstract. A study, on the effects of natural mild fungal infection on meiosis of Bar- ley Pollen Mother Cells (PMCs), was done in order to analyze the chromosomal dam- age elicited by the pathogenic conditions. A pattern similar to common mutagens, of reduction in mitotic index and chiasma frequency, as well as, production of various aberrations that demonstrate chromosomal damage, was observed. The most com- mon abnormalities were un-orientation and other spindle related aberrations, as well as stickiness and clumping of chromosomes. The disease induced a reduction in pol- len viability as compared to the control plants. The results were compared with those of a high dose of a known mutagen ie gamma rays in order to draw commonalities between the two conditions. Keywords: anomalies, Barley, chromosomes, fungal mycotoxins, gamma rays, Puccinia. INTRODUCTION Barley (Hordeum vulgare L.) is among the most important cereal crops in the world. In India, it is used for the purposes of animal feed, flour making and for malting and brewing purposes (Selvakumar et al., 2014; Singh et al., 2019). Barley is a low input crop and has much better adaptability when com- pared to wheat (Verma et al., 2012). Barley is naturally inbred and provides a very good genetic material for study of mutagenesis using various agents like radiations, chemicals or combinations of both. The response of this crop in transferring the mutations from one generation to another is exceptionally good which makes it a preferred choice of material for mutagenic studies. This important crop suffers from various diseases causing great reduc- tion in yield and grain quality. One such disease is the stripe rust caused by Puccinia striiformis f. sp. hordei (Psh). This disease is common in vari- ous countries of South Asia, East Africa, and Central and North America. P.striiformis f. sp. hordei is a macrocyclic rust having two hosts, primary host being Barley. The most damaging spore, in this fungus, is the uredospore which follows multiple asexual cycles to spread the disease. The primary symptoms include yellow/orange pustules (uredosorus) lined linearly along midribs. The diseased plants are shorter, less vigorous and have a poor root http://www.fupress.com/caryologia https://doi.org/10.36253/caryologia-2430 https://doi.org/10.36253/caryologia-2430 https://doi.org/10.36253/caryologia-2430 https://www.fupress.com/caryologia https://www.fupress.com/caryologia https://orcid.org/0000-0002-4510-7974 mailto:viveksingh_2@yahoo.com 14 Vivek Singh system. The photosynthesis is affected as a result of the dark pustules reducing the green area. Grains are poor- ly filled and many florets show abortion. It affects both quantity and quality of grain production (Luthra and Chopra, 1990; Roelfs and Huerta-Espino, 1994). All exogenous agents capable of producing chromo- some aberrations (CAs), i.e., clastogens, are mutagens, and most are also carcinogens. For that reason, cytoge- netic damage has long been a favored surrogate endpoint for assessment of carcinogenic and mutagenic potential. One very important conclusion from ionizing radiation (IR) mutagenesis studies in cells of higher organisms has been that large-scale genomic structural changes gener- ally dominate the spectrum of new mutations, as com- pared with point mutations or other small intragenic changes. Granted, the spectrum of mutations can dif- fer vastly, depending on the mutagenic agent, but in the present study, our emphasis will be on mutagenic events resulting from large-scale structural changes to the host chromosomes genome caused by fungal pathogen- esis. These include deletions, insertions, inversions, and translocations, any of which can disrupt genes, alter the control of gene expression, or even result in expression of new fusion sequences. IR is virtually unique regard- ing its efficiency for producing prompt DNA double- strand breaks (DSBs) randomly throughout the genome, which is the prerequisite lesion for the development of these structural rearrangements (Cornforth et al, 2021). An analogy of the action of IR with fungal toxin will thus enable us to understand the mechanism by which pathogen brings about chromosomal damage and herit- able changes in host. It can be speculated that together with physiologi- cal effects, the pathogens, like fungus, might have some cytogenetic effects on the host plant. Though a num- ber of studies attributing the effect of fungal toxins on inhibition of various enzymes and interference with physiological processes are available the effect at cel- lular and genetic level has not been explored. The level of DNA damage after treatment with fungal metabo- lites would be related to the ability of the host to survive and reproduce after infection. Impaired activity of anti- oxidant defense and DNA repair contribute to the DNA damage by free radicals. A few workers have observed induction of chromosomal anomalies by fungal infec- tions eg Aspergillus on Cotton (El-Naghy, 1992), Fusar- ium on wheat and maize (Helmey, 2003), Fusarium on Maize (El-Daisty, 2009) etc. Various studies have evalu- ated the effects of radiations, chemicals, pesticides, plant metabolites etc on the genetic material for the purpose of mutagenesis but very few studies are available which show the impact of microbial toxins up to the Chromo- somal/DNA level (Kaur et al, 2018). Therefore, it was planned to study the meiosis in fungus infected plants and compare it with a known mutagen ie Gamma radia- tions, in order to evaluate the chromotoxic potential of fungal toxins. MATERIALS AND METHODS Hordeum vugare variety K10 of barley was used for the study. Naturally infected plants were monitored for morphological parameters and young ears collected at the time of flowering. At the flowering time, ie about 50 days after planting, floral buds were collected and fixed in Farmer’s fixative (3:1 absolute ethanol-acetic acid) for 24 h. They were then transferred to 70% alcohol and stored at 4°C. Cytological investigations were done using 1% acetocarmine squash technique. Anaphase and Metaphase stages were considered as active division. All chromosomal abnormalities were screened and recorded under the respective stages of cell division where they occurred. Pollen viability was estimated by Acetocar- mine stain method where deeply stained pollen grains were considered viable, while non-stained ones were considered non-viable. Similarly, gamma irradiated sets of half of LD50 ie 25 kR were screened for comparison of all parameters. Suitable controls were also maintained and all sets given exactly similar environmental condi- tions. RESULTS AND DISCUSSION The control buds showed perfect bivalents at meta- phase I and a separation of 7:7 at anaphase I. Metaphase II and Anaphase II were also perfectly normal in con- trols. However the fungal infected sets showed various types of abnormalities (Fig. 1). The total abnormality percentage was moderate. Common Metaphase anoma- lies included stickiness, clumping, precocious move- ment, fragmentation, multivalent formation, univalents, secondary association, unorientation etc. The Anaphase was also marked with different types of chromosomal anomalies like stickiness, laggards, bridges, unequal sep- aration, multipolarity and micronuclei. Table 1 presents a list of anomalies induced by fungi as well as those induced by gamma rays, on the meiosis of barley. Meiotic anomalies have been reported by a num- ber of workers in a variety of crops following muta- genic treatments eg Wani & Anis, 2008 (Gamma rays on Cicer), Pakorn etal, 2009 (Gamma rays on Anubias), Motilal etal, 2012 (EMS on Astercantha), Akhtar, 2014 15An account of chromosomal damage in PMCs of stripe rust infected barley (Gamma rays and EMS on Solanum), Asare etal, 2017 (Gamma rays on Abelmoschus), Gnankambary etal, 2019 (Gamma rays on Vigna), Chen etal, 2020 (EMS on Ara- chis), Rashid etal, 2021 (Stress on Trillium), Liu et al, 2022 (Natural factors on Elymus), Turkoglu etal, 2023 (Sodium Azide on Triticum) etc. However there are only a few studies which suggest that fungal toxins may also induce chromosomal anomalies. Agar and Alpsoy (2005) studied aflatoxin G1 (AFG1) induced chromosomal aber- rations in Vicia faba and Zea mays. Their results showed that 0.1, 0.2, 0.4 ppm concentrations of aflatoxin G1 could induce gradient based chromosomal aberrations. Since, they point towards the instability of the genome, chromosomal damages and aberrations have often been regarded as the index of cytotoxic potential of a mutagen. As far as radiations and chemical mutagens are concerned, an increase in cytological anomalies is the obvious manifestation. But similar effects induced by mycotoxins, is an interesting aspect of this study. Different authors have given various explanations for occurrence of different anomalies. In general the meta- phase abnormalities are related to spindle dysfunction e.g. scattering, unorientation and precocious movement of chromosomes. An alteration in genes governing spin- dle formation may lead to a loss of directive influence on chromosome arrangement and movement leading to consequent dysfunctional anomalies. The current obser- vations in fungal treatment are in concurrence with stud- ies of Styer and Horace (1984). They treated maize roots with solutions of moniliformin (a metabolite of Fusarium moniliforme Sheldon). They mentioned that higher con- centration caused a disruption of the spindle apparatus. Figure 1. Cytological anomalies induced by chromosomal damage in Barley; 1- Normal Metaphase I, 2 - Normal Anaphase I showing 7:7 separation, 3 – Normal Telophase I, 4 – Stickiness and secondary association of bivalents at Metaphase I, 5 – Multivalents, 6 – Unorienta- tion at Metaphase I, 7 – Clumped multivalents, 8 – Precocious movement, 9 – Fragmentation, 10 – Laggards at Anaphase I, 11 – Chromo- some bridge, 12 – Unequal separation and laggard at Anaphase I, 13 – Precocious movement at Metaphase II, 14 – Bridge at Anaphase II, 15 – Laggards at Anaphase II, 16 – Multipolarity at Telophase II. [Scale Bar 1 cm = 4µ]. 16 Vivek Singh The presence of univalents and multivalents at meta- phase has been reported in different mutagenic studies. Multivalent formation could be attributed to irregular pairing and breakage followed by translocations and inversions. The predominance of ring or chain multi- valents is dependent upon the length of interchanged segments and position of interchange. Stray bivalents at metaphase I and II are usually caused by spindle dis- function (Bhat et al., 2007b). The observed precocious chromosomes migration to the poles may be resulted from univalent chromosomes at the end of prophase I or precocious chiasma terminalization at diakinesis or metaphase I. Precocious migration of univalents to the poles is found to be a very common abnormality among plants which have been treated with mutagens (Conso- laro et al., 1996). Secondary associations can result from modified chromosomes arrangement due to the duplica- tion, interchanges or stickiness (Kumar and Singh 2003). Chromosome stickiness has been reported to be a result of partial dissociation of the nucleoprotein and alteration in their pattern of organization (Evans 1962). Mc Gill et al (1974) and Klasterska et al (1976) suggested stickiness due to improper folding of chromosome fibre. Jayabalan and Rao (1987) reported stickiness in meiosis as due to the disturbances in cytochemically-balanced reactions by secondary effects of radiations. Fragmentations or chromosome shattering observed in present study has also been reported by Cremer and Cremer (1986), Albanese (1982), Cremer et al (1981) as effects of radiation alone or in combination with chemi- cals. These may be due to damaged mechanisms of DNA repair caused by radiations (Periera, 1995). Laggards were one of the most common Anapha- sic abnormalities characterized by delayed movement of some chromosomes during Anaphasic separations. These have been reported by a number of workers and may be due to delayed terminalization, stickiness of chromo- somes ends or because of failure of chromosome move- ment (Permjit and Grover 1985, Jayabalan and Rao 1987, Sohair 1989). These laggards may move randomly to any pole and give rise to unequal separation of chromosomes or they may form a pole by aggregating together and causing multipolarity. These may just clump together while remaining away from daughter nuclei at each pole and form micronuclei at Telophase. Bridges are also a very common chromosome dam- age indicator. These are caused by paracentric inversion, which lead to formation of a dicentric bridge joining two poles (Swanson, 1988). The bridges may also be formed by stickiness between separating chromosomes. During separation these bridges break randomly and give rise to unbalanced poles having unequal chromatin volume. Ta bl e 1. A c om pa ris on o f c yt ol og ic al a bn or m al iti es in du ce d by g am m a ra ys a nd fu ng al in fe ct io n in m ei os is of b ar le y. Tr ea tm en ts C F/ bi v + SE M et ap ha se I/ II a bn or m al iti es (% ) A na ph as e I/ II a bn or m al iti es (% ) Te lo ph as e I/ II a bn (% ) C yt ok i-n es is ab n (% ) TA b (% ) + S E Lm D o Pc M v U v Fg St C l Sa Lg Br U s N s St M p Lg Br M n Tr Pa C on tr ol 1. 66 + 0 .0 5 0. 07 0. 13 0. 20 + 0 .1 1 G am m a Ra ys (2 0 kR ) 1. 48 + 0 .0 6 0. 61 0. 67 0. 33 0. 27 0. 20 2. 03 0. 54 1. 55 1. 01 0. 88 0. 82 1. 21 0. 40 0. 10 0. 81 0. 11 11 .5 4 + 0. 23 Fu ng al in fe ct ed 1. 30 + 0 .1 0 0. 95 0. 36 0. 68 0. 31 0. 29 0. 27 1. 26 0. 82 0. 22 1. 97 0. 77 0. 54 1. 04 0. 47 0. 54 1. 02 0. 41 11 .9 2 + 0. 18 Lm =L at e m ov em en t of b iv al en ts ; D o= D ist ur be d or ie nt at io n of c hr om os om es ; P c= Pr ec oc io us m ov em en t of c hr om os om es ; M v= M ul tiv al en t fo rm at io n; U v= U ni va le nt f or m at io n; Fg =F ra gm en ta tio n of c hr om os om es ; S t= St ic ki ne ss o f ch ro m os om es ; C l= C lu m pi ng o f ch ro m os om es : S a= Se co nd ar y as so ci at io n of b iv al en ts ; L g= La gg in g ch ro m os om es ; B r= Br id ge fo rm at io n be tw ee n po le s; U s= U ne qu al s ep ar at io n of c hr om os om es a t an ap ha se ; N s= N on s yn ch ro no us d isj un ct io n; M p= M ul tip ol ar ity ; M n= M ic ro nu cl ei ; T r= Tr ia ds ; P a= Po ly ad s; TA b= To ta l A bn or m al ity ; S E= St an da rd E rr or o f M ea n. 17An account of chromosomal damage in PMCs of stripe rust infected barley Disturbances in spindle formation in meiosis II leads to formation of three or more than four poles at Ana/Telo- phase II. Subsequent wall formation gives rise to triads or polyads instead of normal isobilateral tetrads. Changes in the surface proteins of PMC walls, lead to clumping of PMCs and sometimes gives rise to cyto- plasmic channels allowing transmigration of chromatin. This is known as cytomixis and it is a powerful agent in causing polyplodization and increase in chromosome numbers within PMCs. Changes in cytoplasmic viscosity may also lead to shrinkage of PMCs, which was evident in a few PMCs. The chiasma frequency showed a decrease in fungi infected plants. Presence of greater number of univalents might be responsible for a consequent decrease in the chiasma frequency although it may get balanced some- what by a simultaneous increase in multivalents. Greater occurrence of rod bivalents might also cause a decrease in chiasma frequency. Some authors like Raghuvanshi and Singh (1974) have reported a decrease in chiasma frequency with increase in dose of treatment while some others like Prasad and Godward (1969) had observed an opposite trend. The reduction observed here is common to most radiation and chemical mutagenic treatments and has been demonstrated by workers like Sinha and Ma hapatra (1969) in Zea, Sinha and Roy (1976) in Pha- seolus and Lal and Srinivasachar (1979) in Pennisetum. A high degree of pollen sterility, in gamma treat- ment as well as fungi infected sets, is a result of increase in the chromosomal abnormalities, which give rise to pollen with varying degrees of chromosomal imbalance. Pollen sterility has been attributed to stickiness that leads to irregular segregation and improper fragmenta- tion of chromosomes. Such unbalanced pollen grains are very often non-viable and unable to fertilize the ovules. This in turn causes adverse impact on seed setting. A comparison of the chromosomal anomalies pre- sent in Fungi infected plants with those present in con- ventional mutagens like gamma irradiated or chemical treated plants reveals a great level of similarity. When we compare the results obtained with by fungal patho- gens with those elicited by other mutagens, we get strik- ing similarities which indicates similar mode of action. Kumar and Yadav (2010) reported almost similar chro- mosomal anomalies induced in Sesamum indicum (L.) by EMS (Ethyl Methane Sulphonate) which is an alkylat- ing agent. Singh et.al (2019) and Nilan et.al (1964) also found identical chromosomal damage was reported by use of radiations. Studies suggest that even some non- conventional agents like Catalase and Lipase enzymes have elicited reduction in germination and survival of plants (Ananthaswamy et.al; 1971). However, if fungal pathogen induced mutations are considered, there was a clear predominance of physiological abnor malities like stickiness and clumping over clastogenic ones like fragmentation or micronuclei. Such anomalies lead to high degree of gamete sterility and bring the plant into a growth dis advantage. As a result high degree of lethality is induced even at low infections. It seems that the reduction in active mitotic division occurs due interference of chemicals in the G1 cell cycle which suppresses DNA synthesis as reported by Mohan- das and Grant (1972) in several higher plants. There are many studies that compare the chromosomal abnormali- ties induced by the chemical, physical mutagens and the combination of both like those of Sree Ramalu (1973), Mehra and Mann (1974), Kumar and Singh (2002), Alam et al(2022) etc. How ever, the progress in the effective and efficient use of mutagens is hindered by complex interplay of many physical and chemical factors that determine the ultimate yield of mutations (Konzak et al 1975). According to Wilson (2019) and Jeong (2014) ion- izing radiations can stimulate ROS production through nitric oxide synthase (NO) pathway. Interaction of NO molecule with superoxide radical (O2 -) to produce per- oynitrite (ONOO-). Peroxynitrite is a powerful oxidant radical reacts with DNA bases, amino acids and lipids. NADPH oxidase is also been reported to cause produc- tion of ROS. When the ROS encounter biological organ- isms, they cause damage to biomolecules such as DNA, RNA and proteins in living cells. 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