Incidence, Characterization and Pathogenicity of Seed-Borne Fungi of Lentil ( Lens culinaris L.) in Pakistan

: Lentil ( Lens culinaris Medik.) is an important protein-rich Rabi pulse crop after chickpea in Pakistan. Its area and production in the country is decreasing drastically due to its susceptibility to various biotic and abiotic stresses. Among biotic-stresses, seed-borne fungi are the most imperative issue and the accurate identification and characterization of target pathogen isolates/races is crucial for the management of plant diseases in lentils. Therefore, the current study was performed to identify the fungi associated with lentil seeds, to confirm their pathogenicity and to assess their incidence. For this purpose, isolation of fungal pathogens was made on potato dextrose agar medium (PDA) from 56 Lens culinaris L. genotypes. These pathogens were characterized using morphological and molecular techniques and their pathogenicity was performed following blotter paper and agar plate method. Seven fungi belonging to five genera were isolated from lentil seeds ( Fusarium avenaceum , Alternaria alternata, Aspergillus flavus, Aspergillus niger, Aspergillus tubingensis , Penicillium citrinum and Bipolaris sorokiniana ) of different genotypes. Of these seven fungal species, F. avenaceum , A. tubingensis and B. sorokiniana are new records for lentil seeds from Pakistan. Aspergillus flavus was found to occur in high frequency followed by A. niger , F. avenaceum , P. citrinum , A. alternata , A. tubingensis and B. sorokiniana . Under pathogenicity test on blotter paper and on PDA F. avenaceum , A. flavus, A. niger, A. tubingensis were highly pathogenic, while A. alternata, P. citrinum and B. sorokiniana were found to be less pathogenic. The presence of well-known toxigenic fungal pathogens in lentil seeds suggests the possible risk of contamination of the seeds and enhances the possibility of pre-and post-infections of crop. Therefore, the present study will help to devise effective management strategies to reduce contamination in seeds and also to control the further spread of these pathogens to reduce crop losses.


INTRODUCTION
Lentil (Lens culinaris L.) belongs to the family Fabaceae and is one of the oldest quick-cooking and nutritious staple-legume Rabi crops.It is cultivated in more than 70 countries but extensively grown in Canada, Turkey, Syria, Nepal, Bangladesh, India and China.It is normally grown in sandy loam soil and is drought resistant.Lentil can also be grown in water logged and saline soils (Hussain et al., 2007).It is a quick-cooking and nutritious staplelegume which is consumed globally in complete, dehulled, and split form (Khazaei et al., 2019).About one-third of the calories in lentil come from protein, which is the third-highest level of protein by weight of any pulse crop.In many parts of the world, lentil is the cheapest protein food and contains dietary fibre, vitamin B, minerals, iron and important amino acids (lysine, arginine, and leucine) (Khazaei et al., 2019).Lentil seeds are lens-shaped with a broad range of seed coat colours (green, tan, brown, gray, white and black) and pattern (marbled, dotted, spotted, complex, and un-patterned) (Khazaei et al., 2019).
Lentil (Lens culinaris Medik.) is an important Rabi pulse crop after chickpea in Pakistan which contains about 25% protein (Zia-Ul- Haq et al., 2011) and plays an important role in cereal-based diet of the common man.Being a leguminous crop, it restores soil fertility through biological nitrogen fixation and increase seed yield from 23-25% and straw yield up to 16% (Muscolo et al., 2014).The demand of this protein-rich pulse is increasing every year and will continue to feed the ever-rising population of the country.Although its demand is increasing every year, its area and production in the country is declining rapidly as compared to past decades.During the past decade the area under lentil crop has drastically reduced which is an alarming situation.World production of lentil is primarily coming from India (36%), Canada (17%) and Turkey (15%).Other neighbouring countries of Pakistan, like Nepal (14%), Bangladesh (3%) and Iran (3%) also have a much higher share by producing 165, 119 and 115 thousand tonnes respectively, when compared to Pakistan (7.25 thousand ton) (Sarwar et al., 2014).The reduction in production still continues and multiple factors may be involved in the low production which needs to be addressed to meet the country's demand and to cut short imports to the country.To increase production, it is important to increase its cropping area and this is only possible if we evolve better genotypes that can replace poor yield potential and less resistant varieties.Among other reasons of low production in the country, one is non-availability of quality seed of high yield potential varieties having inbuilt resistance against abiotic and biotic stresses along with wide adaptability.
to nearly half of it (12,923 hectares) in 2019 (Anonymous 2021a).According to economic survey of Pakistan (2010Pakistan ( -2020) ) (Anonymous 2021b), the production of Lentil remained the same of last year's production, and out of 51 major lentil producing countries, Pakistan stands at 49 th position with percentage share of only 0.1% to lentil global area and production (Anonymous 2021c).
The yield of lentils in Pakistan is generally poor due to its susceptibility to various stresses biotic and abiotic and existing plant architecture.Among biotic stresses a number of diseases caused by fungi, bacteria, viruses, nematodes and phanerogamic plant parasites are major constraints.These are responsible for the deterioration of seed quality, seed rots, seedling mortality and stem and root diseases.These diseases reduce the vigour of the plant, threatening its life and ultimately minimizing yields.These diseases may cause complete crop failure under favourable conditions, and disease development and can be the major limiting factor for lentil cultivation in certain areas.
Seed health plays a vital role in successful cultivation and yield of a crop species.A literature survey showed that many fungal species have been reported from lentil seeds.Among these, species of the genera Fusarium (wilt), Sclerotinia (watery soft-rot), Rhizoctonia (wet root rot), Macrophomina (dry root rot) and Pythium (damping-off) are economically important seed and soil-borne pathogens causing wilt and root rot complex (Makkouk et al., 2003).Seed borne infection not only cause seed rot/decay and germination failure but it is also an important survival mechanism for fungi.More importantly, it spreads the fungus into disease free areas and contaminates the soil.These diseases are very difficult to control through the use of costly chemicals or cultural practices and even the recovery of heavily infected and damaged plants is impossible.Therefore, the cultivation of resistant varieties is the only viable, safe and economical control measure for these diseases.
Comprehensive understanding of pathogen populations and diversity at the species level is important, as high genetic diversity indicates a rapid change in the genetic structures.This, in turn, shows the development of more virulent species and strains in response to management practices, changed environments, and increased biological fitness of these species.So far, no systematic study on the population and pathogenicity of seed-borne pathogens associated with lentil seed has been conducted in Pakistan using both morpho-cultural and molecular tools.Therefore, Considering all the above, this study has been proposed with the objectives; i) to isolate the seed-borne fungi associated with different lentil genotypes; ii) to assess the incidence of isolated fungal pathogens; iii) to identify the seed-borne fungi associated with different lentil genotypes using morphological and molecular techniques and iv) to confirm the pathogenicity of isolated fungal pathogens.

Collection of seed sample
Lentil seed samples belonging to 56 different lentil genotypes were collected from Plant Breeding and Genetics division, Nuclear Institute for Agriculture and Biology (NIAB), Faisalabad.These seeds were harvested manually and stored at room temperature in cloth bags in laboratory of Plant Pathology at NIAB, Faisalabad for six months.No fungicides were applied to these seeds during storage.

Isolation of fungal pathogens from Lentil seeds
The isolation of seed borne fungal pathogens from lentil seeds were made on potato dextrose agar (PDA) medium.Firstly 35-40 seeds of each test genotype were surface sterilized with 3% sodium hypochlorite for 10 min.These seeds were then thoroughly washed with sterilized distilled water twice to remove the toxic effect of sodium hypochlorite and air dried on sterilized blotter papers for 30 minutes.Then five sterilized seeds were placed on 90 mm petri-plate containing PDA medium.These petriplates were then incubated for 4-5 days at 25±2 °C under 12h, alternating cycles of artificial day light and darkness in an incubator (Rahim et al., 2010).Then the growing hyphal tips were further transferred on the petri-plates for purification and then on slants for identification and preservation for future work.Morphological identifications were made with the aid of literature (Thom and Rapper, 1945;Raper and Thom, 1949;Barnett and Hunter, 1972;Booth, 1971;Ellis, 1971 and1976).Isolation percentage of the fungal isolates recovered from different genotypes of lentil was calculated by using the formula of Spurr and Wetly (1972)

Molecular characterization of fungus
Total genomic DNA was extracted from a seven-dayold culture of each test fungi isolated from lentil seeds for molecular characterization using a modified CTABbased DNA extraction method (Doyle, 1987).Its internal transcribed spacer (rDNA-ITS) region was customsequenced (Eurofins Genomics, USA) using primers pair ITS-1/4, Tef-1F and Tef-1R and Bt2F and Bt4R.The attained sequences were analyzed with the BioEdit software.BLASTn (http://blast.ncbi.nlm.nih.gov/Blast.cgi) searched for comparison and a consensus sequence was submitted to GenBank (http://www.ncbi.nlm.nih.gov/genbank/).Reference (rDNA-ITS) region sequences of different fungal spp.were retrieved from GenBank for phylogenetic reconstruction and were aligned via MUSCLE (Edgar, 2004).Phylogenetic analysis was conducted using MEGA7 (Kumar, 2016) and a tree was constructed using the maximum likelihood method (ML) based on Tamura-Nei model (Tamura and Nei, 1993) with 1000 pseudo-replicates.

Pathogenicity test
Pathogenicity test of the isolated seed-born fungi of lentil was conducted in laboratory using following two methods.

Blotter method
For the determination of pathogenicity on blotter paper, Maham Sajjad et al. seeds of each test genotype were first surface sterilized in 3% sodium hypochlorite solution for 10 minutes and washed in autoclaved distilled water three times and then air dried properly (Vidić et al., 2013).Then spore suspension of each isolated seed-born test fungi was prepared by adding autoclaved distilled water in a beaker containing seven days old fungal culture of each test fungi isolated from lentil seeds on PDA.The spore suspension was then filtered through a muslin cloth to remove mycelial debris and adjusted to 1 × 10 6 conidia per ml using a haemocytometer.Then seeds of each test genotype were dipped in spore suspension of each fungus separately for 30 minutes.Ten seeds of each genotype treated with fungal suspension were transferred on three-layer sterilized distilled water soaked filter papers in 140 mm diameter sterilized petri-plates.There were three replicates for each treatment.The plates were then incubated at 25±2°C for 12hrs of alternating cycles of day/night under fluorescent light in an incubator.More sterilized distilled water was added as needed to prevent seeds from drying out and to allow seeds to germinate.Seeds were examined daily and data was collected as germination percentage (5 th day post inoculation), seedling mortality percentage (15 th day post inoculation), infection percentage (IP), infection type range (ITR), disease severity index (DSI) and disease response (DR) on the basis of DSI value ( Brown lesions affecting 76-100% of the hypocotyl and or radicle followed by complete necrosis and or wilting 3.5-4.4Susceptible

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Seeds completely covered by fungus followed by no germination 4.5-5.0Highly susceptible both plumule and radical) but not produced a healthy plant.Data was recorded three times within a week following the rating system described by Leisso et al., (2011) with modifications.

Agar plate method
For the determination of pathogenicity on PDA each test fungi isolated from lentil seeds was cultured on 14 mm diameter sterilized petri-plate for seven days (Yli-Mattila et al., 2018).Seeds of each test genotype were surface sterilized in 3% sodium hypochlorite solution for 10 minutes and washed in autoclaved distilled water three times and then air dried properly.Ten seeds per petri-plate of each test genotype were then transferred on PDA medium containing the test fungus.There were three replicates for each treatment.The plates were then incubated at 25±2°C for 12 hour of alternating cycles of day/night under fluorescent light in an incubator.Seeds were examined daily and data was collected as germination percentage (5 th day post inoculation), seedling mortality percentage (15 th day post inoculation), infection percentage (IP), infection type range (ITR), disease severity index (DSI) and disease response (DR) on the basis of DSI value (Table 1; Figure 1).Seedling mortality percentage was calculated by the percentage of viable seed germination (produced both plumule and radical) but not produced a healthy plant.Data was recorded three times within a week following the rating system described by Leisso et al., (2011) with modifications.

RESULTS
Under present study seven species of fungi belonging to five genera were found to be associated with seeds of fifty-six lentil genotypes.Following cultural, microscopic and molecular characteristics, these fungal species were identified as Fusarium avenaceum (Fr.)Sacc., Alternaria alternata (Fr.)Keissler, Aspergillus flavus Link, Aspergillus niger Van Tiegh, Aspergillus tubingensis Mosseray, Penicillium citrinum Thom and Bipolaris sorokiniana (Table 2).
The summary of incidence of fungi associated with the seeds of lentil is presented in Table -3.Of the tested two- A PCR amplification of about 600bp of was achieved using the internal transcribed spacer (ITS) universal primers.
Fusarium avenaceum was isolated from twenty-two genotypes A. flavus was isolated from twenty-nine genotypes, A. niger was isolated from thirty genotypes, A. tubingensis from one genotypes, P. citrinum from two genotypes, A. alternata from two genotypes and B. sorokiniana from on genotype (Table 3; Table 4).As a whole no genotype was reported as highly resistant or resistant but two genotypes were designated as moderately susceptible, eight were susceptible and two were highly susceptible (Table 6).

Pathogenicity of Aspergillus flavus
Seeds of all genotypes inoculated with spore suspension of A. flavus showed 100% germination on blotter paper.No (0%) seedling mortality percentage was observed for eleven tested genotypes while 13.3% was reported for one genotype M-85 whereas minimum as 13.3% for two genotypes viz.As a whole no genotype was reported as highly resistant or resistant but one was reported as moderately resistant, one moderately susceptible and remaining ten were designated as susceptible (Table 5).
Disinfected seeds of all genotypes showed varying degree of germination when directly placed on PDA containing 7 days old culture of A. tubingensis.Maximum germination percentage (30%) was observed for two genotypes namely Punjab Masoor-2019 and Punjab Masoor-2020.However, no germination was observed for four genotypes namely M-85, NIAB Masoor 2006, NIA Masoor 05 and M-93.Infection percentage was reported as 100% while ITR ranged from 3-5 for all genotypes (Data not shown).All genotypes were designated as highly susceptible as DSI value was ranged from 4.6 to 5.0 for these genotypes (Table 6).

Pathogenicity of Aspergillus niger
On blotter paper maximum germination percentage as 100% was reported in case of seven genotypes (M-  DSI value was reported as 3.6 for genotype Punjab Masoor-2009 and designated as highly susceptible while minimum as 1.0 for Punjab Masoor-2019 and designated as resistant.On the basis of DSI value two genotypes were designated as resistant, three as moderately resistant, four genotypes were moderately susceptible and three were susceptible (Table 5).
Disinfected seeds of all genotypes showed varying degree of germination when directly placed on PDA containing 7 days old culture of A. niger.Maximum germination percentage (70%) was observed for genotype Punjab Masoor-2019, however, no germination was observed for four genotypes (Shiraz-96, Punjab Masoor-2009, Mansehra-89 andNIAB Masoor-2006).IP was reported as 100% while ITR ranged from 3-5 for all genotypes (Data not shown).One genotype was designated as susceptible with 3.9 DSI value and eleven as highly susceptible with DSI value ranging from 4.5-5.0(Table 6).

Pathogenicity of Penicillium citrinum
Seeds of all tested genotypes inoculated with spore suspension of P. citrinum showed 100% germination on blotter paper following no seedling mortality in all genotype.IP was reported as 6.7% for genotype M-85 and 20.0% for Punjab Masoor-2009 andPunjab Masoor-2019 with ITR value ranged from 1-2 for these genotypes (Data not shown).No infections were observed for the remaining nine genotypes and were ranked as highly resistant.However, DSI value for genotype M-85 was observed as 0.1, for Punjab Masoor-2009 as 1.4 and for Punjab Masoor-2019 as 0.2 and ranked as resistant (Table 5).
Disinfected seeds of all genotypes showed 100% germination when directly placed on PDA containing 7 days old culture of P. citrinum furthermore, no seedling mortality was observed in all the tested genotype.Maximum infection percentage was reported as 33.3% for genotype M-85 and minimum as 21.4% for NIAB Masoor-2002 with ITR value ranged from 1-3 for these genotypes (Data not shown).No infections were observed for the remaining ten genotypes and were ranked as highly resistant.However, DSI value for genotype M-85 was observed as 2.0 and for NIAB Masoor-2002 was as 2.3 and was ranked as moderately resistant (Table 6).

Pathogenicity of Alternaria alternata
Seeds of all tested genotypes inoculated with spore suspension of A. alternata showed 100% germination on blotter paper followed by no seedling mortality for all the tested genotype (Data not shown).No infection was reported in any tested genotype and all were ranked as highly resistant (Table 5).
Disinfected seeds of all genotypes showed 100% germination when directly placed on PDA containing 7 days old culture of A. alternata except for one genotype M-93 that showed 90% germination.Seedling mortality was 11.1%, IP was 20%, ITR value was 4-5 (Data not shown) and DSI value was 4.5 for genotype M-93 and was designated as highly susceptible.However, no seedling mortality, infection percentage and DSI value was reported for the remaining eleven genotypes and were designated as highly resistant (Table 6).

Pathogenicity of Bipolaris sorokiniana
Seeds of all the tested genotypes inoculated with spore suspension of B. sorokiniana showed 100% germination on blotter paper followed by no seedling mortality for all the tested genotype (Data not shown).No infection was reported in any tested genotype and all were ranked as highly resistant (Table 5).
Disinfected seeds of all genotypes showed 100% germination when directly placed on PDA containing 7 days old culture of B. sorokiniana (Data not shown).No infection was reported in any tested genotype and all were ranked as highly resistant (Table 6).

DISCUSSION
A healthy seed of any crop is the most significant contribution for crop production as it is the basis of a healthy plant, an essential condition for excellent yield.Amongst a variety of issues which influence the seed health, the most imperative is seed borne fungi that can cause decrease in seed germination and seed vigor.Lentil seeds in storage bear a mycoflora of field and storage fungi.Fungi are the major group of the seed-borne pathogens owing to their ability of reproduction and continued existence in environment.Field fungi steadily vanish and storage fungi then prevail.That may be the basis of discoloration of the seeds, germination failure and ultimately also resulting in loss of market value.These also give rise to numerous unwanted alterations making them unfit for consumption and planting.Therefore, the control of seed-borne fungi is very important to minimize the harmful effects of these pathogens (Narayan et al., 2013).To control any disease accurate identification of involved pathogen is the basic requirement.The pathogens are constantly changing in nature and the accurate identification and characterization of target pathogen isolates/races is crucial for the management of plant disease.Relatively little work has been done in Pakistan and worldwide using morphological and molecular techniques on lentil seed-borne diseases.Conventional fungal taxonomy has become a powerful and more authenticated by the aid of molecular biology and integration of computational biology as well.Taking into account the significance of lentil crop, the current study was performed to study the association of microflora with the seeds of advanced genotypes of lentil using morphological and molecular techniques followed by their pathogenicity studies.A crop becomes contaminated with fungi during the course of harvesting, accumulation, and transfer.During this process storage fungi grow vigorously and start seed spoilage and produce numerous mycotoxins.Mycotoxins are toxic secondary metabolites produced by numerous fungal species.Aspergillus species can produce aflatoxins and ochratoxin A, Fusarium species can produce deoxynivalenol, zearalenone, fumonisins, HT-2 and T-2 and Penicillium species can also produce ochratoxin A (Kumar et al., 2021(Kumar et al., & 2022)).These toxins are dangerous to human being and animals.
In the current study seven fungi belonging to five genera were isolated from lentil seeds (F. avenaceum, A. alternata, A. flavus, A. niger, A. tubingensis, P. citrinum and B. sorokiniana).Of these seven fungal species, F. avenaceum and A. tubingensis are new records from Pakistan for lentil seeds.Aspergillus flavus was found to occur in high frequency in the seeds of tested lentil genotypes.Our findings agree with the earlier results of Hussain et al. (2007) and Rahim et al. (2010), they found that the A. flavus was the most dominant fungus on lentil seeds.Molecular analysis showed that A. flavus isolate recovered from lentil seeds in this study have 100% similarity with formerly reported isolates CP051048 (recovered from Zea mays in USA), CP051080 (recovered from Zea mays in USA), MT328475 (recovered from sand and seawater), MH063950 (recovered from complementary food in Nigeria), MH063942 (recovered from complementary food in Nigeria), MH063947 (recovered from complementary food in Nigeria), MH063944 (recovered from complementary food in Nigeria), CP082259 (recovered from Rhipicephalus micoplus eggs in Mexico).Aspergillus flavus is a universal filamentous mold that can grow under diverse environmental conditions (saprophytic and pathogenic) and is known to produce the most toxigenic strains of aflatoxins (B1 and B2) in food crops.Under pathogenicity test, seeds of all genotypes inoculated with spore suspension of A. flavus showed 100% germination on blotter paper with no seedling mortality except for one genotype M-85.Minor seedling infection was observed for five genotypes and seven were infection free.However disinfected seeds of all genotypes showed varying degree of germination (40-80%) when directly placed on PDA containing culture of A. flavus.Seedling mortality was also observed as 25-100 followed by DSI value from 2.6-4.6 in these genotypes.This shows that both pathogenicity test methods were working well but PDA method was found comparatively better for evaluation of lentil seed pathogenicity against A. flavus.
Aspergillus niger was detected in 30 lentil genotypes with 3.6 percent seed infection.Our findings agree with the earlier results of Hussain et al. (2007) and Rahim et al. (2010), they found that the A. niger was the most common fungus on tested lentil seeds.Molecular analysis showed that A. niger isolate recovered from lentil seeds in this study have 100% similarity with formerly reported isolates AJ853742 (recovered from Australia), MK336494 (recovered from Ipomoea pes-caprae in Taiwan), MN069571 (recovered from subterranean termites Reticulitermes grassei in Southern Spain), MN788114 (recovered from Cocoa in Nigeria), MT447518 (recovered from roots of Lycium barbarum L. in China) and MT316340 (recovered from Compost pile in Italy).The fungus A. niger is cosmopolitan and normally found in the soil, existing as a saprophyte on rotting vegetation and foliage, compost heaps, stored grains and as well as a contaminant of food.Aspergillus niger is reported to produce fumonisin mycotoxins B2 and B4.Toxigenic strains of A. niger can also produce Ochratoxins in stored grains and livestock feed.Ochratoxins can be transferred into milk in human beings, rabbits and rats, and also blood and internal organs of livestock that fed on polluted feed (Dania et al., 2021).Under pathogenicity test, on blotter paper 100% germination was reported in case of seven genotypes followed by 93.3 % in case of four genotypes and 80% in case of one genotype followed by seedling mortality as 46.7% in case of two genotypes while 20% in case of one genotype Mansehra-89.IP was reported as 100% for eleven genotypes except one genotype Punjab Masoor-2020 that showed 50%.Following DSI value two genotypes were designated as resistant, three as moderately resistant, four genotypes were moderately susceptible and three were susceptible.Furthermore, disinfected seeds of all genotypes showed varying degree of germination and IP value when directly placed on culture of A. niger and one genotype was designated as susceptible and eleven as highly susceptible.This shows that both pathogenicity test methods were working well but PDA method was found comparatively better for evaluation of lentil seed pathogenicity against A. flavus.
Fusarium avenaceum was first time detected in 22 lentil genotypes with 3.0 percent seed infection in Pakistan.Our findings agree with the earlier results of Abdel-Hafez (1984) and Morrall et al. (2001) who isolated F. avenaceum for the first time from lentil seeds in Saudi Arabia and Canada.F. avenaceum is a worldwide distributed filamentous ascomycete fungus generally present in soil, seed and a wide range of plants that can infect every plant tissues.As a result F. avenaceum can affect crop production and cause considerable yield reductions and economic losses to farmers.It can be capable of producing toxic metabolites that are harmful to humans and livestock during use of contaminated foods.Phytotoxic effects of F. avenaceum mycotoxins include seed decay, prohibition of seed germination, root and shoot development during plant growth.It can produce disease symptoms like damping off, wilting, chlorosis, necrosis and fruit and root rots.Spores of F. avenaceum might be spread by contaminated seeds, air currents and by fungal gnats (Pollard and Okubara 2019).F. avenaceum was first described in 1886 (Yli-Mattila et al., 2018).However, in lentils root rot caused by F. avenaceum was first time documented in eastern Washington (Lin and Cook 1977).Under pathogenicity test, seeds of all genotypes inoculated with spore suspension of F. avenaceum showed 100% germination on blotter paper except for one genotype LNUYT-1902 following 100% seedling mortality for one genotype LNUYT-1902 and 20% for genotype Shiraz 96 while no for remaining.Disinfected seeds of all genotypes showed varying degree of germination when directly placed on PDA containing F. avenaceum culture following 100% germination and seedling mortality for six genotypes.This shows that both pathogenicity test methods were working well but PDA method is found comparatively fine for evaluation of lentil seed pathogenicity against F. avenaceum.Our findings are in line with the previous findings of Lin and Cook, (1977), Fletcher et al. (1991), Hwang et al. (1994) MH614525 and MH614529 (Belgium).Previous A. tubingensis has never been reported from lentil in any disease perspective.A. tubingensis is found everywhere in the world and can grow on fresh parts as well as mainly on dead plant matters.It is morphologically very alike to black fungus A. niger, due to which it is much hard to recognize them based only on morphological characteristics.Though, A. tubingensis might microscopically be able to be differentiated by its production of seclorotia that shows a white to pinkish color characteristic.A. tubingensis have the capacity to produce ochratoxin, a very hazardous toxin to animal and human health.It is also found to cause skin and lungs diseases.It has been found to infect agronomically significant food crops by damaging their foliage and fruits.A. tubingensis also has the ability to manufacture many mycotoxins which are injurious to poultry, livestock, fish and human being (Khizar et al. 2020).Under pathogenicity test, seeds of all genotypes inoculated with spore suspension of A. tubingensis showed 100% germination on blotter paper except for two genotypes followed by varying degree of seedling mortality in all genotypes.However, under PDA test no germination was observed for four genotypes followed by 10-30% germination in other genotypes.Seedling mortality was also 100% except for one genotype and all genotypes were designated as highly susceptible.This shows that both pathogenicity test methods were working well but PDA method is found comparatively fine for evaluation of lentil seed pathogenicity against A. tubingensis.Our findings agree with the earlier results of Alomran et al. (2020), they reported for the first time that A. tubingensis is a seed-borne pathogen of date palm and can affect seed germination and seedling emergence of date palm.
Penicillium citrinum was detected in 02 lentil genotypes with 0.2 percent seed infection.Our findings agree with the earlier results of Hussain et al. (2007) and Rahim et al. (2010), they found that the P. citrinum was found to be associated with lentil seeds as seed-borne pathogen.Molecular analysis showed that P. citrinum isolate recovered from lentil seeds in this study have 100% similarity with formerly reported isolates KX674625 (recovered from indoor air in turkey), MH858380 (recovered in Netherlands), LT558884 (recovered from clinical samples in USA), LT558885 (recovered from clinical samples in USA), LT558887 (recovered from clinical samples in USA) and LT558889 (recovered from clinical samples in USA).The fungus P. citrinum has been reported from soils, leaves, stems, fruits, seeds, sporecases (sporangia) of fern and pollen of cereals, fruits, legumes etc. and indoor environments (Houbraken et al. 2010;Ashtekar et al. 2022).P. citrinum is known to consistently produce a mycotoxin named as citrinin with strong nephrotoxicaction.Under pathogenicity test seeds of all tested genotypes inoculated with spore suspension of P. citrinum showed 100% germination on blotter paper and no seedling mortality was observed in all genotype.Infection was reported as 6.7% for genotype M-85 and 20.0% for Punjab Masoor 2009 and Punjab Masoor 2019.On PDA having culture of P. citrinum disinfected seeds of all genotypes showed 100% germination no seedling mortality.Moderate infection was reported on two genotypes namely M-85 and NIAB Masoor 2002 showing that lentil genotypes are less susceptible to P. citrinum.
Alternaria alternata was detected in 02 lentil genotypes with 0.1 percent seed infection.Our findings agree with the earlier results of Hussain et al. (2007) and Rahim et al. (2010), they found that the A. alternata was found to be associated with lentil seeds as seed-borne pathogen.Molecular analysis showed that A. alternata isolated from lentil seed have the 85.4% similarity with the previously reported isolate MK248606 (recovered from Phragmites australis in Egypt) and MN548784 (recovered from Cherry fruit in China).A. alternata is a well-known facultative fungal-pathogen of a number (over 380 host plant species) of economically significant plants world-wide.This fungus is known to cause leaf blight, brown leaf spots, post harvest fruit rot and as seed-borne pathogen of many plants.A. alternata is everywhere in the atmosphere and is also reported from indoor grown plants.Its spores are sources of natural contamination and effective allergens and the Alt a1 protein was found to be the major cause of fungal allergies in human-beings.A. alternata was reported to fabricate more than 70 phytotoxins, and exposure to A. alternata in houses was found to be completely linked with asthma (Wachowska et al. 2021).A. alternata can survive in harsh conditions and can pass the winterweather in the soil, seed, infected plant debris or perennial host plant tissue including bark, nodes and scaly leaves in the form of mycelia, conidia and as resting structures to defy the adverse conditions (Troncoso-Rojas and Tiznado-Hernández 2014).Under pathogenicity test seeds of all tested genotypes inoculated with spore suspension of A. alternata showed 100% germination on blotter paper and on PDA containing culture of B. sorokinian followed by no seedling mortality and no infection except for one genotype M-93 that showed 90% germination followed by 11.1% seedling mortality and 20% infection.
Bipolaris sorokiniana was detected in 01 lentil genotypes with 0.04 percent seed infection.Our findings agree with the earlier results of Iftikhar et al. (2009) and Kouadri et al. (2021), they found that the B. sorokiniana was pathogenic to lentil.This fungus is soil and seed-borne and can infect a number of host plants belonging to several genera and families like wheat, barley, other cereals and grasses etc. Lentil was earlier reported as an experimental host of B. sorokiniana in Pakistan (Iftikhar et al. 2009) and as leaf spots pathogen of lentil in Algeria (Kouadri et al. 2021)).It can cause diseases like common root-rot, spot-blotch, and black-point.In wheat seeds spores of this fungus can remain viable for 10 years and as a resting mycelium for 5 years Al-Sadi (2021).The teleomorph (perfect state) for B. sorokiniana is Cochliobolus sativus (Ito & Kuribayashi) Drechs.ex Dastur.However, C. sativus was not documented in nature, except in Zambia.Though, sexual reproduction of C. sativus has been infrequently documented.Moreover, the reproduction of B. sorokiniana generally takes place through the production of asexual-conidia (Al-Sadi, 2021).Under pathogenicity test seeds of all tested genotypes inoculated with spore suspension of B. sorokinian showed 100% germination on blotter paper and on PDA containing culture of B. sorokinian followed by no seedling mortality and no infection.This shows that B. sorokinian was not pathogenic on lentil seeds but may be primary source of leaf spot disease of lentil.
Under pathogenicity test variable level of resistance/ susceptibility were observed between same genotypes for the blotter paper and PDA test.This may be due to the level of inoculum applied to seed.Under blotter paper method quantity of inoculum delivered to the seed was limited while in case of PDA a permanent source of inoculum was present.

CONCLUSION
The current study showed that different fungal pathogens (F. avenaceum, A. alternata, A. flavus, A. niger, A. tubingensis, P. citrinum and B. sorokiniana) are associated with lentil seeds.It was also observed that lentil genotypes showed differences in susceptibility to seed-borne fungal contamination.The presence of these fungal pathogens with lentil seeds may prove destructive and can enhances the possibility of pre-and post-infections of crop under both field and storage conditions.Therefore, the present studies will help to devise effective management strategies to control the further pre-and post-infections spread of these pathogens to reduce crop losses due to agricultural soil contamination by these fungi.

Figure 1 :
Figure 1: Photographic representation of disease severity rating scale used for the evaluation of pathogenicity of fungi isolated from lentil seeds under controlled conditions.

Figure 5 :
Figure 5: The Phylogenetic relationships between present and other related isolates and species of Aspergillus (Aspergillus flavus and A. tubingensis) based on the internal transcribed spacer region of rDNA (ITS).Related sequences were obtained from GenBank.

Figure 6 :
Figure 6: The Phylogenetic relationships between present and other related isolates and species of Aspergillus niger based on the internal transcribed spacer region of rDNA (ITS).Related sequences were obtained from GenBank.

Figure 7 :
Figure 7: The Phylogenetic relationships between present and other related isolates and species of Penicillium based on the internal transcribed spacer region of rDNA (ITS).Related sequences were obtained from GenBank.

Figure 8 :
Figure 8: The Phylogenetic relationships between present and other related species of Alternaria based on the internal transcribed spacer region of rDNA (ITS).Related sequences were obtained from GenBank.

Table 1 :
Disease severity rating scale used to evaluate the of pathogenicity of fungi isolated from lentil seeds under controlled conditions.

Table 2 :
Cultural, microscopic and molecular characteristics of different fungi isolated from lentil seeds on potato dextrose agar medium.

Table 3 :
Percentage of the lentil seeds infected with different seed-borne fungi.

Table 4 :
Isolation of different fungi from seeds of different varieties of lentil.

Table 6 :
Pathogenicity test of different fungal pathogen (isolated from lentil seed) on PDA against different lentil genotypes (NIAB Masoor-2002 and ChakwalMasoor while minimum as 20% in case of genotype Mansehra-89.IP was reported as 100% for eleven genotypes except one genotype Punjab Masoor-2020 that showed 50%.ITR ranged from 1-5 for all tested genotypes (Data not shown).Maximum ; DR= Disease response; HR= Highly resistant; R= Resistant; MR= Moderately resistant; Moderately susceptible; Susceptible; HS= Highly susceptible

Table 5 :
Pathogenicity test of different fungal pathogen (isolated from lentil seed) on blotter paper against different lentil genotypes DR= Disease response; HR= Highly resistant; R= Resistant; MR= Moderately resistant; Moderately susceptible; Susceptible; HS= Highly susceptible Maham Sajjad et al.
and Zitnick- Anderson et al. (2021), they found that F. avenaceum is pathogenic to lentil.Aspergillus tubingensis was detected in one lentil genotypes with 0.04 percent seed infection.Molecular analysis showed that A. tubingensis isolate recover from Maham Sajjad et al.