Seed Health Testing Research Articles

Prevalence of Seed-Borne Fungi on Soybean (Glycine max L. Merr.) Seeds Stored Under Medium-Term Cold Room Facilities: Implications for Genebanks

This study examined the prevalence of seed-borne fungi in polyethylene plastic-packaged soybean seeds stored in a genebank and identified factors influencing their incidence. Twenty-four seed lots were sampled from the collection stored at 10 °C in the World Vegetable Center genebank in Arusha, Tanzania. The seeds used were those regenerated and harvested in 2015, 2016, and 2017. A seed health test was conducted for sterilized seeds on potato dextrose agar, sterilized seeds on top of the paper, and unsterilized seeds on top of the paper. Seven-year-old sterilized seeds plated on top of the paper exhibited the highest germination percentage (74 ± 7.09%) and lowest fungal incidence (7.00 ± 4.41%). Conversely, seven-year-old unsterilized seeds plated on top of the paper had the lowest germination (22.00 ± 6.97%) and highest fungal incidence (79.00 ± 8.31%). Older seed lots showed significantly higher equilibrium seed moisture content (Eqmc), though seed age had no significant influence on germination percentage or fungal incidence. Seed germination percentage showed a significant negative relationship with Eqmc, though Eqmc had no significant effect on fungal incidence. Meanwhile, germination percentage showed a significant negative relationship with fungal incidence. Six fungi isolates were identified using their morphological features from soybean seed samples: Absidia glauca, Aspergillus niger, Fusarium spp., Mucor hiemalis, Pestalotiopsis versicolor, and Sordaria macrospora. It is concluded that high fungal incidence negatively affects seed germination but is not significantly correlated with seed moisture content. The dry and cold storage conditions in the genebank, while effective in extending seed longevity, can unintentionally allow seed-borne mycoflora to persist. Although fungi may not actively proliferate under these conditions, they may do so when favorable conditions are restored, such as during germination tests, and consequently may compromise seed viability. Therefore, this study emphasizes the importance of increased systematic seed health checks before storage in genebanks to ensure optimal seed quality, and the replacement of all polyethylene plastic bags to aluminium foil packaging.

Phytotoxic effect of Carica papaya extract on seed-borne fungus of African Yam Bean (Sphenostylis stenocarpa Hochst ex. A. Rich. Harms) seeds

Extracts of many higher plants have been reported to exhibit antifungal properties under laboratory experiments. This study was therefore carried out to investigate the effect of Carica papaya plant extracts on the African Yam Bean seed-borne pathogen. The seed health test of African Yam Bean was carried out using the blotter paper method. The experiment was conducted at the Plant Pathology Laboratory of the Department of Crop Science and Horticulture, Nnamdi Azikiwe University, Awka. Test of plant extract for inhibition of radial growth of seed-borne pathogen of African yam bean was studied under in vitro experiment at 0%, 50%, 75% and 100% concentrations with 0% as the control. The design was a Completely Randomized Design (CRD) with three replications. The test plant extracts of different concentrations were added into Petri dishes containing molten-sterilized Sabouraud Dextrose Agar (SDA) and swayed gently on the laboratory bench to allow an even mixture These were allowed to gel. Then nine millimeter discs of a seven-day pure culture of Aspergillus flavus were aseptically placed on the centre of the Petri dishes containing the SDA-extract mixture. Records on the radial inhibition effect of the test plant extract were kept for further analysis. Data on radial inhibition of fungus were subjected to Analysis of Variance (ANOVA). Results showed that Aspergillus niger, Aspergillus flavus and Aspergillus terreus were isolated from incubated African Yam Bean seeds. Carica papaya leaf extract used was very effective and the higher the concentrations of extract, the more effective in the inhibition of radial growth of the test fungus. It could therefore be recommended that farmers should always conduct viability tests on procured seeds before planting. Farmers should rather use plant extract such as the one used in this experiment in controlling seed-borne fungal pathogens of African Yam Bean seeds than synthetic fungicides.

Development and validation of a multiplex real‐time RT‐PCR test for the screening of tomato (Solanum lycopersicum) for the detection of pepino mosaic virus

AbstractPepino mosaic virus (PepMV) is known to cause a wide range of symptoms in cultivated tomato plants, damaging fruits and crops. Currently, five genotypes of PepMV are known (US1, CH2, EU, LP and PES), which can all infect tomato plants. Tomato seed is a proven pathway for PepMV, and the virus is known to easily spread mechanically during crop handling. A multiplex real‐time reverse transcriptase‐polymerase chain reaction (RT‐PCR) test was developed and validated for the specific identification of PepMV isolates from tomato seeds. This test is designed to be used for diagnostics and seed health tests as a pre‐screen to distinguish negative PepMV samples from suspect samples or as a confirmation test of a positive ELISA or bioassay test result. The multiplex real‐time RT‐PCR test consists of two PepMV‐specific real‐time RT‐PCR tests and a Bacopa chlorosis virus real‐time RT‐PCR test as an internal amplification control. The analytical specificity of the developed test was evaluated by in vitro and in silico analyses. The analytical sensitivity was evaluated by creating three dilution series of naturally PepMV‐infected leaf tissue in healthy tomato seed extracts and the diagnostic specificity and sensitivity were validated on a collection of seed lots.

Aggressive Alternaria brassicicola with Reduced Fungicide Sensitivity Can Be Associated with Naturally Infested Broccoli Seeds.

Alternaria brassicicola is a part of the Alternaria complex that causes leaf blight and head rot (ABHR) in brassica crops. Infested broccoli seeds can play an important role in introducing A. brassicicola in transplant houses and production fields. However, characterization of natural seed infestation and seed-to-seedling transmission of A. brassicicola in broccoli is yet to be demonstrated. In this research, we characterized Alternaria spp. isolates from commercial broccoli seedlots for their species identity, pathogenicity, and aggressiveness on broccoli and their sensitivity to a quinone-outside inhibitor (QoI) fungicide (azoxystrobin). Two hundred commercial seedlots from two broccoli cultivars, Cultivar 1 (EC; n = 100 seedlots) and Cultivar 2 (ED; n = 100 seedlots) were, evaluated for the presence of A. brassicicola under in vitro conditions using a seedling grow-out assay. Alternaria spp. was detected in 31 and 28% of the commercial seedlots of Cultivar 1 and Cultivar 2, respectively. The seed-to-seedling transmission (%) varied considerably within each positive-infested seedlot, which ranged from 1.3 to 17.3%. Subsequent molecular identification of single-spore cultures (n = 138) was made by sequencing four housekeeping genes: actin, the major allergen (Alta1), plasma membrane ATPase, and glyceraldehyde-3-phosphate dehydrogenase (GPD), and the sequences were concatenated and compared for the phylogenetic distance with diverse Alternaria species. Ninety-six percent (n = 133) of the isolates formed a cluster with a known A. brassicicola based on a multigene phylogeny, which were later confirmed as A. brassicicola using a species-specific PCR assay. One hundred percent of the A. brassicicola seed isolates (n = 133) were either highly or moderately aggressive on broccoli (cultivar Emerald Crown) based on a detached leaf assay. Sensitivity of representative A. brassicicola isolates (n = 58) to azoxystrobin was evaluated using a spore germination assay, and the EC50 values (effective fungicide concentration [ppm] at which germination of conidia of isolates were reduced by 50% compared to control) for each isolate was determined. A. brassicicola isolates from naturally infested commercial broccoli seeds were sensitive to azoxystrobin with considerably low EC50 values in the range of <0.0001 to 0.33 ppm; however, there were a few isolates (14%) that showed 100-fold reduced sensitivity from the most sensitive isolate (EC50 = 0.0001 ppm). Our results confirm that commercial broccoli seedlots can be naturally contaminated with pathogenic and aggressive A. brassicicola. We also provide evidence for the potential presence of A. brassicicola isolates with reduced azoxystrobin-sensitivity in naturally infested commercial broccoli seedlots, which has never been reported before. Together, these findings may have implications in considerations for seed-health testing, seed treatments, and greenhouse scouting to limit introduction of infested seedlots in commercial broccoli fields.

Seed Health Testing of Recombinant Inbred Lines Derived from Cross between Kalonunia and Pusa Basmati 1637

Seed Health Testing of Recombinant Inbred Lines Derived from Cross between Kalonunia and Pusa Basmati 1637

Hyperspectral imaging for the detection of plant pathogens in seeds: recent developments and challenges.

Food security, a critical concern amid global population growth, faces challenges in sustainable agricultural production due to significant yield losses caused by plant diseases, with a multitude of them caused by seedborne plant pathogen. With the expansion of the international seed market with global movement of this propagative plant material, and considering that about 90% of economically important crops grown from seeds, seed pathology emerged as an important discipline. Seed health testing is presently part of quality analysis and carried out by seed enterprises and governmental institutions looking forward to exclude a new pathogen in a country or site. The development of seedborne pathogens detection methods has been following the plant pathogen detection and diagnosis advances, from the use of cultivation on semi-selective media, to antibodies and DNA-based techniques. Hyperspectral imaging (HSI) associated with artificial intelligence can be considered the new frontier for seedborne pathogen detection with high accuracy in discriminating infected from healthy seeds. The development of the process consists of standardization of methods and protocols with the validation of spectral signatures for presence and incidence of contamined seeds. Concurrently, epidemiological studies correlating this information with disease outbreaks would help in determining the acceptable thresholds of seed contamination. Despite the high costs of equipment and the necessity for interdisciplinary collaboration, it is anticipated that health seed certifying programs and seed suppliers will benefit from the adoption of HSI techniques in the near future.

Seed-borne fungi: challenges in seed health testing for biosecurity and agricultural sustainability

Seed-borne fungi: challenges in seed health testing for biosecurity and agricultural sustainability

Characterization of Seed-to-Seedling Transmission of Alternaria brassicicola in Broccoli.

Alternaria brassicicola is part of a complex of Alternaria species that causes leaf blight and head rot in brassica crops such as broccoli, kale, cabbage, cauliflower, and collards. Seed can serve as a potential source of inoculum for the transmission of A. brassicicola in broccoli as demonstrated earlier; however, seed-to-seedling transmission of pathogen was never characterized empirically. So, the objectives of this study were to (i) re-evaluate the effect of artificial seed infestation on seed germination and seed-to-seedling transmission of A. brassicicola in broccoli; (ii) determine the effect of A. brassicicola-seed inoculum levels on seed-to-seedling transmission; (iii) evaluate if variations in A. brassicicola aggressiveness affect A. brassicicola seed-to-seedling transmission; and (iv) evaluate seed treatments that can reduce seed-to-seedling transmission of A. brassicicola in broccoli. Artificially infested seedlots were generated by inoculating broccoli seeds with a spore suspension of 1 × 105 conidia/ml of A. brassicicola using the vacuum infiltration method. Inoculated (n = 10 seedlots; 300 seeds/seedlot) or control seedlots in three replicates were planted on two layers of sterile blotter paper saturated with sterile water in transparent plastic boxes and incubated at 20°C and >90% relative humidity (RH) under continuous fluorescent light. Percent seed germination and percent seed-to-seedling transmission were recorded every other day for 21 days. Percent seed germination was significantly affected with artificial pathogen inoculation. One hundred percent of the seedlots transmitted the pathogen to broccoli seedlings, and seed-to-seedling percentages of the seedlots varied considerably. A strong linear and significant relationship between A. brassicicola inoculum level and seed-to-seedling transmission (%) within each seedlot was observed. Interestingly, variations in aggressiveness of A. brassicicola isolates did not affect seed-to-seedling transmission, as 100% of the seedlots were able to transmit the pathogen. Seed treatment with Miravis (a.i. pydiflumetofen 18.3%) significantly increased seed germination and reduced seed-to-seedling transmission percentages in A. brassicicola-inoculated seedlots. These results indicate that artificial seed inoculation with A. brassicicola can result in consistent seed-to-seedling transmission with significant impact on seed germination. Seed inoculum density of ≥104 conidia/ml is necessary for reliable transmission of A. brassicicola. Further seed-to-seedling transmission is not dependent on aggressiveness of A. brassicicola isolates and seed treatment with Miravis can significantly reduce pathogen transmission in broccoli seedings. Overall, this study provides detailed characterization of seed-to-seedling transmission of A. brassicicola in broccoli that can be further used to determine inoculum threshold, which has potential applications in seed-health testing and sample size determination. Furthermore, we also provide options for effective seed treatments that can significantly reduce A. brassicicola seed-to-seedling transmission and may potentially aid in managing seedborne fungal infection.

Chapter 7: Seed health testing

Chapter 7: Seed health testing

Identification on seed associated mycoflora of sesame genotypes

Seed mycoflora of 165 genotypes of sesame in (AVTs &amp; IVTs) were detected using seed health testing methods viz., paper towel method, standard blotter method, and standard agar plate method and six distinct fungi viz.,Alternaria, Macrophomina, Fusarium, Aspergillus, Penicillium, and Rhizopus spp. were detected. In the three methods employed to identify seed mycoflora the standard blotter method has identified the maximum number of seeds infected by fungi compared to paper towel method and standard agar plate method. After a few days of germination, it was observed that the majority of the seeds were infected with disease, which hampered normal seedling growth and resulted in abnormal and rotted seedlings.

Quantification of Plant Virus Seed Transmission Rate in Arabidopsis thaliana.

More than 25% of all known plant viruses are transmitted through seeds, which makes this mode of dispersal of great importance for plant virus epidemics. Virus detection in seed stocks remains the most frequent approach for seed health testing, but current methods are not always standardized and/or do not allow analyzing large numbers of seeds. Here, we describe a high-throughput method to quantify plant virus seed transmission rate based on classical grow-out tests, which can be applied to widely different viruses and host species.

Perlakuan Air Panas dan Plant Growth Promoting Rhizobacteria untuk Menekan Cendawan Terbawa Benih pada Padi Varietas IPB-3S

Hot Water Treatment and Plant Growth Promoting Rhizobacteria to Suppress Seed-Borne Fungi on Rice Variety IPB-3S&#x0D; IPB-3S is a new rice variety which was developed to improve national rice production in Indonesia. This study aimed to determine seed-borne fungi associated with IPB-3S variety and to determine the potential of hot water and PGPR treatment to suppress the fungal infection. The study consisted of five experiments, i.e. seed health test by dry seed observation and by blotter test, pathogenicity test, seed treatment using hot water and plant growth promoting rhizobacteria (PGPR). Seven genera of fungi were found in IPB-3S seeds, namely Aspergillus, Penicillium, Rhizopus, Rhizoctonia, Curvularia, Fusarium and Colletotrichum. All of these fungi are pathogenic based on pathogenicity test. Seed treatment using hot water at 54 ℃ and PGPR reduced fungal infections and increased germination of IPB-3S seeds.

Development and validation of real‐time PCR tests for specific identification of Xanthomonas species causing bacterial spot disease on tomato (Solanum lycopersicum) and pepper (Capsicum annuum)

AbstractBacterial spot is a significant disease of tomato and pepper crops caused by members of four Xanthomonas pathogens from three species, namely Xanthomonas euvesicatoria pv. euvesicatoria, Xanthomonas euvesicatoria pv. perforans, Xanthomonas vesicatoria and Xanthomonas hortorum pv. gardneri. These Gram‐negative bacterial plant pathogens are recommended for regulation as quarantine pests by the European and Mediterranean Plant Protection Organization and are currently regulated on tomato and pepper seeds in many regions of the world. Tomato and pepper seeds are a transmission pathway for all three species. Real‐time PCR TaqMan tests were developed and validated for specific identification of isolates from tomato and pepper seeds of these pathogens. These tests are designed to be used in seed health tests after isolation on semiselective media to reduce the number of isolates that need testing in follow‐up pathogenicity tests. A second set of real‐time TaqMan PCR tests was subsequently developed targeting two markers for each taxonomic group of strains. The analytical specificity of all developed tests was evaluated with in vitro and in silico analyses. Diagnostic specificity and sensitivity were also validated on collections of isolates in several laboratories.

Seed Borne Fungal Pathogens Associated with Finger Millet Accessions

Seed health testing to detect seed-borne pathogens is an important step in the management of crop diseases. A study on the detection and identification of seed-borne fungi was done using seeds of finger millet (Eleusine coracana (L.) Gaetrn) accessions collected from different parts of the country. The research was undertaken to determine different seed-borne fungal pathogens associated with seeds of finger millet accessions from November 27, 2020 to February 25, 2021 at National Agriculture Genetic Resources Centre, Khumaltar, Lalitpur. A total of 85 seed samples, collected from different parts of the country, were used for the research. A hundred seeds of each sample were tested for the presence of seed-borne fungi using the Deep Freeze Blotter method following the International Rules for Seed Testing Association, 2001, and identification was done based on the growth habit, and morphology of the spores observed. One or more pathogens were detected in 65 out of 85 samples. Four different pathogens, namely Bipolaris nodulosa, Alternaria spp., Cladosporium spp., and Pyricularia grisea were identified. B. nodulosa and Cladosporium spp. were predominant in most of the samples with seed infection in 42 (49.4%) and 40 (47.05%) seed accessions, respectively. Hence, it is highly recommended to undertake focused research on the economic significance of the seed-borne fungi, while considering their impact and effective management strategies in order to enhance seed health management.

Focus Issue Articles on Diagnostic Assay Development and Validation: The Science of Getting It Right

Focus Issue Articles on Diagnostic Assay Development and Validation: The Science of Getting It Right

Seed Health Testing: Doing Things Right

Since seeds can be a route for pathogen introduction, they are routinely inspected and tested to prevent pest outbreaks and introduction into new territories. The need for high throughput, short lead times, and cost reduction has played an important role in the development and application of techniques in seed health testing. Examples are molecular and serological techniques, such as ELISA and PCR assays, which are commonly called indirect tests. After signal detection in ELISA or PCR assay a seed lot is a suspect lot that requires further investigation for a final conclusion about the health status of the seed lot, since these tests do not provide any information about pathogen viability or pathogenicity. The seed industry uses them as a prescreen to identify healthy seed lots and in combination with classical methods, commonly called direct tests, to confirm viability of the target pathogen and demonstrate its pathogenicity. However, outside industry, indirect tests are increasingly used to make a final decision on the health status of a seed lot. This has led to a growing number of seed lots being rejected when the risk of introducing a pathogen to importing countries may have been negligible. We propose that investments continue to be made in the development of high-throughput prescreening detection methods like HTS and PCR assays, but together with direct tests that enable accurate assessment of the risks involved when target pathogens are detected using indirect tests. Close collaboration between molecular scientists and classical phytopathologists is essential. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

DETEKSI DAN IDENTIFIKASI JAMUR PATOGEN TERBAWA BENIH VARIETAS PADI LOKAL DI KABUPATEN KAPUAS

This study was aimed to detect and identify seed-borne pathogenic fungi in six local rice varieties of Siam Unus, Karangdukuh, Mayang, Gumpal Kuning, Gumpal Putih, and Siam Arjuna in Kapuas Regency. In addition, the objective of this study was also to determine the effect of seed pathogens on the physiological quality and the percentage of infected seeds.&#x0D; The research location was at the Agricultural Cultivation Laboratory, Faculty of Agriculture, University of Palangka Raya, and the study was carried out from September 2020 to February 2021. Seed health testing used the filter method (blotter test), isolation and identification of seed-borne pathogenic fungi using the agar method. The variables observed included macroscopic morphology of pathogens in petri dishes and microscopic evaluation using a compound microscope, seed germination, percentage of infected seeds, frequency of microorganism findings and pathogenicity tests on seed germination.&#x0D; Based on the results of the study, the identified pathogenic fungi carried by local varieties of rice seeds in Kapuas district were Aspergillus spp., A. flavus, A. niger, Curvularia sp. and Oidium sp., however these pathogens did not affect seed germination. The highest numbers of microorganisms observed on the agar method was Aspergillus spp. (37.5%), followed by Curvularia sp. (29%), whereas in the blotter test method the frequency of the fungus Curvularia sp. only 5.6%. The frequency of finding A. flavus and A. niger on the agar method was lower at 21.8%, while the fungus Oidium sp. was detected in very low percentage for only 0.9%. The pathogenic fungus Oidium sp. showed the highest level of pathogenicity in seeds reaching 21.7%, while the pathogenic A. niger, Curvularia sp. and A. flavus showed a lower infection rate, at 13.9% to 17.8% respectively. These pathogenic fungi might cause the seeds to not germinate normally such as leading to necrosis of the hypocotyl and plumules, stunted germination growth, and death of seed germination.&#x0D; Keywords: Detection, identification, seed-borne pathogens, local rice varieties, Kapuas

First report of Diaporthe helianthi infecting safflower: A threat to its cultivation in India

AbstractDuring seed health testing of 160 accessions of safflower, we observed some fungal growth on seed surface of four accessions i.e., IC337954, IC338197, EC182199 and EC181301 which were received from Hyderabad, Telangana, India for long‐term conservation in the National Genebank, ICAR‐National Bureau of Plant Genetic Resources, New Delhi, India. The associated fungus was morphologically identified as Diaporthe helianthi. Later on, the identity was confirmed as Diaporthe helianthi (Desm.) Grove by the sequencing of internal transcribed spacer (ITS) region (GenBank Id: KM979906.1). During seed health testing, seed infection of 30.0 to 70.0 per cent was recorded with no seed germination in infected seeds. Previous findings revealed that D. helianthi is host‐specific pathogen causing stem canker to sunflower only and the fungus is seed‐borne. As a destructive pathogen causing substantial yield losses, especially to sunflower, it may be a serious threat to safflower cultivation in the country and anywhere else. Therefore, the detection and identification of D. helianthi can be helpful in alarming the threat at an early stage thereby minimizing the probable damage to safflower cultivation in near future.

First report of Diaporthe phaseolorum infecting Indian trumpet flower (Oroxylum indicum) from India

Seed health testing, using the blotter method, revealed some fungal growth on the seed surface of one accession of Indian trumpet flower/Broken bones tree (Oroxylum indicum (L.) Kurz) collected from Kokrajhar, Assam, India. The fungus was identified as Diaporthe phaseolorum (Cooke &amp; Ellis) Sacc. based on morphological characters. Later, the identity was re-confirmed by DNA sequencing using ITS gene sequencing (NCBI Sequence Id: MT154253.1) and a large subunit of rRNA (NCBI Sequence Id: OL798081.1). Literature reveals that D. phaseolorum is a destructive pathogen causing severe yield losses in various host crops. However, detection of D. phaseolorum in Indian trumpet flower seed followed by pathogenicity on its seedlings confirms that O. indicum is a new host record. Being a destructive pathogen of several other crops, such as seed decay and stem canker in soybean, it may pose a serious threat to future cultivation of this herbal plant.

BIOCIDAL-EFFICACY OF GORGONEIA LATIFOLIUM, MYRISTICA FRAGANS AND ALPRON- PLUS ON SEED HEALTH, GERMINATION PERCENTAGE, SEEDLING VIGOUR OF SWEET MELON (CUCUMIS MELO L.) SEEDS AND ASSOCIATED FUNGI ORGANISMS

Laboratory experiment was conducted to test the effects of bio-medicinal plants (Gongronema latifolium (Utazi) leaves and Myristica fragans (Nutmeg) seeds) and synthetic fungicide (Alpron- Plus) on seed health, germination percentage and seedling vigour of sweet melon seeds. The experiment was conducted at the Crop Science and Horticulture Laboratory, Nnamdi Azikiwe University Awka, Anambra State. Bio-medicinal plants were used as crude powdery seed dressers as well as synthetic fungicide. The experiment was laid out in a simple completely randomized design (CRD) with ten replications which consisted of blotter paper germination/ seed health tests of sweet melon seeds and seedling vigour and seedling vigour index tests using soil medium. Ten petri dishes and ten polybags were used respectively for each of the plant extracts and synthetic fungicides simultaneously for both blotter paper and soil medium tests. Twenty seeds which were independently dressed with various plant extracts and synthetic fungicide were plated into one 9cm Petri dish containing three layers of blotter paper in circular form. For soil medium method, ten polybags with few small perforations underneath filled with sterilized top soil were used for each fungicide. The Petri dishes containing the plated seeds were incubated on the Laboratory benches for eight days while seedlings in polybags were kept in the screenhouse and observations were taken and recorded daily for germination count, fungal growth then germination index and seedling vigour index which were derived later with appropriate formulae and their values being expressed in percentages. The results of this investigation revealed that bio-medicinal plant crude extracts had significant effect on germination percentage and germination index of Sweet- melon seeds. Gongronema latifolium crude extract had the highest germination index. The synthetic fungicide was effective in protecting the seeds of sweet melon though had low germination percentage. Nutmeg was not effective when used in crude powdery form as dressers as it had the lowest germination percentage and the highest infestation percentage. It could therefore be recommended that the plant extracts tested in this experiment should not be used as powdery seed dressers but as essential oil using appropriate extracting solvents in order for the bioactive ingredients to act effectively in preventing micro-organisms that hinder germination of sweet melon seeds.