Incidence of seed borne fungi in farm saved rice seeds, quality declared seed and certified seed in Morogoro Region in Tanzania

Rice ( Oryza sativa L.) is the second most important food crop after maize in Tanzania. It faces many challenges like diseases, pests and physical damages, which reduce the seed quality. This article identified microorganisms and the district where they occurred. 13 fungal species comprising of 11 pathogenic and 2 saprophytic fungi were detected and the incidence level varied among sample. Mvomero District showed the highest incidence recorded (64.5%) for sample collected from Hembeti and Dakawa/Msufini villages. In Kilombero, the fungal specie which detected highest incidence was Fusarium equiseti (31%) followed by Fusarium moniliforme (28.5%). Verticillium cinnabarinum and Curvularia inaequalis had the least incidence of 1 and 1%, respectively. The study recommends that before planting season, seeds should be tested to minimize spread of fungal species.


INTRODUCTION
Rice (Oryza sativa L.) is a globally important staple food for more than half of the world's population (Khush et al., 2013).The crop is increasingly becoming an important staple food and cash crop in Africa (Tanko et al., 2016).In this continent, 15 million tons of rice is produced annually (Ronald et al., 2014).Eastern and Southern Africa contributes 16.1% of rice where the major contributors are Madagascar and Tanzania (Food and Agriculture Organization (FAO), 2013; United Republic of Tanzania (URT), 2012).
In Tanzania, rice is the second most important and popular food crop after maize (URT) The major rice producing regions in Tanzania include Morogoro, Shinyanga, Mbeya, Mwanza, Rukwa and Tabora, respectively.The trend of increase or decline of rice yield in Tanzania has not been clear for the past 20 years, due to numerous factors including emergence and poor management of production.Rice yields may fluctuate *Corresponding author.E-mail: agneskwaloe1985@gmail.com.Tel: +321886622032; +231776669440.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License depending on the effects of climate change, poor agricultural practices, and inadequate standard of harvest and post-harvest techniques deployed.
Seed is one of the three basic elements for crop production and help to increase agricultural productivity as it provides the maximum limit of crop yield for all other production inputs.Unlike fertilizers and pesticides, farmers cannot produce without seed (Miva et al., 2017).
However, seeds carry pathogens such as fungi, bacteria, nematodes and viruses responsible for transmitting seed-borne diseases, which often cause partial or total crop losses (Barret et al., 2015).When seed has good physical, physiological, health and genetic qualities, farmers have greater prospects of producing a good crop (Miva et al., 2017).
The present investigation has been carried out to establish baseline information on quality of farm saved rice seed by smallholder farmers, to enhanced strategy of yield improvement.This is important as, many farmers' uses own stored seeds for the next cropping season, though seed borne diseases can be transferred easily and hence other quality attributes may be sub-standard.Proper assessment of the quality of seed, stored by farmers from their previous crop will establish the broad picture of seed quality of locally produced rice as compared to Quality Declared Seed (QDS) and Certified Seed.Therefore, this paper aims to identify the seed borne fungi and evaluate their incidence in farm saved rice seeds in Morogoro Region in Tanzania.

Description of the study area and collection of rice samples for laboratory tests
The rice samples were collected from 45 farmers, 15 from each district in the region.Laboratory investigations were carried out from mid-December 2017 to June 2016 at the African Seed Health Centre and Tanzania Official Seed Certification Institute (TOSCI) by Sokoine University of Agriculture (SUA) in Tanzania.
Multi-stage and cluster-sampling techniques were used to identify the village samples, which ensure good representativeness of rice farming population in the study areas.Rice seed samples of 1.5 kg were collected from each farmer packed in a paper bags, labeled and transported to the African Seed Health Laboratory at Sokoine University of Agriculture then stored in the refrigerator at 5°C (to avoid further development of microorganisms), for laboratory test.

Isolation and identification of the fungi
Seedborne fungi collected from rice sample were detected by Blotter method (Yu et al., 2015).Sterile blotter papers with 9 cm diameter were placed on the Petri dishes.200 seeds were evaluated from each sample.8 Petri dishes is used per sample.Twenty five seeds placed in each Petri dish in a radial manner on the blotter papers were incubated at 25°C for 24 h, of an alternating cycle light and darkness for 7 days.After 7 days, seeds were examined using a stereomicroscope to determine the presence or absence of fungal growth.
Fungal conidia and conidiomata were detected by light microscope.Various magnifications were used to identify conidia Kwaloe et al. 163 and mycelia produced by each group of fungi.Individual genus was classified to species level, using respective keys (Senbeta and Abdella, 2014).The referenced two hundred seeds were used to determine the incidence of fungal microorganisms.Fungal species, found growing on the surface of seeds were identified and their incidence was calculated as:

Data analysis
Difference in the various samples, representing pathogens and types of sources of seeds (Farmer Saved Rice, certified and (QDS)) were established, through mean separation using Turkey's test after significant ANOVA results at P≤ 0.05.Correlation among the incidence of different microorganisms detected during health test was also analyzed using Viera et al. (2016) method.

Fungal incidence of seeds infestation per district surveyed
Table 2 gives a summary of the fungal incidence of seeds infestation per district surveyed.Sample seeds observed were all infested with fungal at different level.3000 rice seeds were analyzed from each district for the presence of, seed-borne microorganisms.Mvomero district recorded the highest percentage of contaminated seeds (16.36%), followed by Kilosa District (13.10%) and the lowest in Kilombero District (12.43%).The highest number of fungi species detected was 10, 9 and 8 in Kilosa, Kilombero and Mvomero, respectively.

Conidia of identified fungal species; the most prevalent species
Characteristics of the identified fungal species and conidia of few species are summarized in Table 2 and Plates 1 to 3. The characteristics generally ranged from, appearance of colony (colour or shape) and morphology of species structures under microscope.Plates 1, 2 and 3 further illustrate conidial characteristics detected.

Incidence =
No .ofrice seeds on which fungus appear Total No .ofseeds examined × 100

Correlation analysis
Correlation analysis coefficients among physical quality attributes and seedborne fungi incidences, were observed between Curvularia lunata and F. moniliforme Results from the current study also revealed significant correlation between seed physical quality and fungi incidences (Table 3).There was a positive correlation between germination and C. lunata Moisture content was also correlated with F. pallidoroseum (0.347*), C. inaequalis (0.353**), Aspergillus (0.360*) and Penicillium (0.35).Conversely, fungi incidence did not affect seed purity, with exception of A. alternata.

DISCUSSION
Seedborne fungi observed in this study are usually found in rice seeds, as compared to study conducted by Tokpah et al. (2017).In additional, Azam et al. (2012) reported a rate of 13 to 20% for A. alternate, 10 to 17% for F. moniliforme and 8.4% for Curvularia spp. in rice seeds.Sharma and Kapoor (2016) obtained similar results.In this study, there were many seedborne fungi in rice seeds, as compared to report of Wang et al. (2012).
The highest rate of incidences of seedborne fungi in rice seeds was found in Mvomero, followed by Kilosa.These districts have a wide range of seed varieties compared to Kilombero.Other comparable reasons might be storage practices and seed handing, which might lead to high incidence level.High incidences include F. moniliforme (65%) and F. equiseti (31%) with C. lunata (16.5, 20.5, and 23%) which may have justifications.It is also important that, these fungal species are commonly found in rice seeds worldwide despite the locations and climatic conditions (Azam et al., 2012).
Frequent occurrences of seedborne fungi in farmer's saved seeds were due to the fact that, most seeds are stored in the rooms which result to difficulty in temperature and humidity control.The environmental factors such as temperature, moisture and relative humidity affect the growth of seed-borne.Generally, high temperature of 15 to 30°C is required for growth and survival of many fungi in the seeds and high relative humidity of more than 65% is required for spore germination (Singh et al., 2012).
F. moniliforme, which causes Bakanae disease and Brown spot on rice seeds have negative impact on the quality of rice (Singh et al., 2012) and are commonly detected.Since farmers in the study area did not report these diseases as constraints, the species do not cause much yield loss in rice.It was reported that, Brown spot disease in rice cause losses of 3 to 15% of yield which is lower than loss caused by rodents and birds.Owolade et al. (2011) determined that, cereal crops including rice seeds colonized by fungal species during storage were responsible for low germination and

Table 1 .
Fungi incidence in rice seed samples detected from Morogoro Region.

Table 2 .
Summary of fungal species and their incidence on farmer saved rice seed collected from rice growing districts in Morogoro Region*.

of contaminated seeds Seed contamination (%) No. of detected species Most prevalent species
*The number of samples were 15 and the number of tested seeds by District were 3,000 in Kilombero, Kilosa and Mvomero.Number in bracket means the first (1), second (2) or third (3) most prevalent among samples from each districts.Plate 1. Conidia of Bipolaris oryzae under microscopy (× 750 mignification).Plate 2. conidia of Fusarium monliforme under microscopy (× 40 magnification).