Genetic Diversity of Colletotrichum sublineolum Isolates from a Single Field in Southern Ethiopia and Evidence for the Existence of MAT2 Genotypes in Different Parts of the Country

An aflp analysis was carried out to study the genetic diversity of Colletotrichum sublineolum isolates collected from a single field in southern Ethiopia. The aflp analysis revealed the presence of genetic variation among the tested isolates. Dice similarity coefficient ranged from 0.69 to 0.96 averaging at 0.87. Cluster and principal coordinate analyses categorized the isolates into nine and six major groups, respectively. The presence of diverse isolates even in a single field should be given due consideration in future breeding programs. Such a diversity calls for a need to stack diverse resistance genes using the gene pyramiding technique to come up with durable sources of resistance against C. sublineolum. A mating type genes identification study was also conducted with the help of PCR on 23 single spore isolates of C. sublineolum collected from different parts of Ethiopia. The degenerate primers SKCM1 and NcHMG were used to amplify the mating type (mat) genes, mat1 and mat2, respectively. mat2 genes were amplified from 15 of the 23 isolates while no amplification product was obtained using skcm1. This may suggest that Ethiopian C. sublineolum populations might be composed of only mat2 genotypes. However, further work that includes large number of isolates from several sorghum growing regions is needed to come up with a conclusive result on the mating system of the pathogen populations in Ethiopia

Spatial Distribution of Enset Bacterial Wilt (Xanthomonas campestris Pv. musacearum) and its Association with Biophysical Factors in Southwestern Ethiopia

አህፅሮት የእንሰት አጠውልግ በሽታ በዛንቶሞናስ ካምቴስትሪስ ፓቶቫር ሙሳሴረም በሚባል የባክቴሪያ ተህዋስያን አማካኝነት የሚከሰትና በኢትዮጵያ ውስጥ እንሰትን ከሚያጠቁ በሽታዎች ውስጥ ቀዳሚውን ስፍራ የያዘ አደገኛ በሽታ ነው፡፡ የዚህ ጥናት አላማ በደቡብ ምዕራብ እንሰት አብቃይ አካባቢዎች ላይ የዚህን በሽታ ስርጭትንና በሽታው በአካባቢው በሚለዋወጡ ከሥነ-ሕይዎታዊና ሥነ-አካላዊ ነገሮች ጋር ያለውን ዝምድና ለማጥናት ነው፡፡ ለዚህም በ10 ወረዳዎች ውስጥ በሚገኙ የ120 የእንሰት ማሳዎች ውስጥ የዳሰሳ ጥናት ተካሂዷል፡፡ በበሽታው የተጠቁ የእንሰት መጠን (Incidence) በአማካይ በወረዳዎች መካከል 23.67- 31.92 በመቶ ነው፡፡ እንደዚሁም የበሽታው የጥቃት መጠኑን በተመለከተ (Severity) ከፍተኛ የሆነ ጥቃት (62.50 በመቶ) በሰሜን ቤንች ወረዳ የተመዘገበ ሲሆን በአንድራቻ ወረዳ ዝቅተኛ የጥቃት መጠን (49.58 በመቶ) ተመዝግቧል፡፡ ሎጅስቲክ ሪግሬሽን ሞዴል እንደሚያስረዳው የእንሰት አጠውልግ በሽታ ስርጭቱ ከ25 በመቶ በላይ ሆኖ የመገኘት አጋጣሚው የእንሰት ማሳ የአፈሩ pH ከ5.5 - 7 መሆን፣ የእንሰት ሰብል ብቻ በአንድ ማሳ ውስጥ መትከል፣ በሽታ ተቋቋሚ ያልሆነ የእንሰት ዝርያ፣ ዝርያውን ከሌላ አርሶ አደር ማሳ መጠቀም እና ምንም ዓይነት አረም ማረምና የበሽታ መከላከያ ያልተካሄደበት ማሳ ናቸው፡፡ በሌላ በኩል ደግሞ የበሽታ ጥቃት መጠን ከ55በመቶ በላይ የመሆኑ አጋጣሚ የአየር ፀባይ የሰሜን-ቤንችንና የየኪ ወረዳዎችን የሚመስሉ አካባቢዎች፣ አረም በሚታረምበት ወቅት እንሰትን በገጀራ መቁረጥ፣ ተቋቋሚ ዝርያ ያልሆነ የእንሰት ተክል፣ የእንሰቱ ዕድሜ ከመካከለኛው እስከ ምርት ለመስጠት ያለው ጊዜ፣ የአርሶ አደሩ ለበሽታው ያለው ዝቅተኛ ግንዛቤ ናቸው፡፡ የዚህ ዳሰሳ ጥናት ግኝቶች እንደሚያመለክቱት የእንሰት አጠውልግ በሽታ በከፍተኛ ሁኔታ በደቡብ ምዕራብ ኢትዮጵያ አካባቢዎች ላይ የተሰራጨ ሲሆን የስርጭቱን መጠን ለመቀነስ እንሰትን እንደ አንድ አማራጭ የማሳው አፈሩ pH ከ5.5 እስከ 7 መካከል ውጪ መትከል፣ ከሌላ ሰብል ጋር አቀላቅሎ መትከል፣ በእንሰት ማሳ ውስጥ የምንጠቀምባቸውን የመገልገያ መሳሪያዎችን ማፅዳትና መጠቀም፣ በበሽታ የተጠቃውን ከማሳው ነቅሎ ማውጣትና ማቃጠል፣ የባለሙያ ምክር መጠቀም መቻል እና በአርሶ አደሮች መካከል የእንሰት ዝርያ መለዋወጥ እንዳይኖር  ማድረግ ናቸው፡፡             Abstract Enset (Ensete ventricosum) bacterial wilt (EBW), caused by Xanthomonas campestris pv. musacearum, is one of the highly destructive diseases of enset  in Ethiopia. Field survey was conducted to determine the distribution of EBW and its association with biophysical variables in Southwestern Ethiopia. In the survey, 120 enset fields in 10 major enset growing districts were assessed. The mean disease incidence across districts ranged from 23.67 to 31.92%, and significantly different levels of disease severity were recorded among districts. Thus, among districts, the highest mean disease severity of 62.50% was recorded from Semen-bench, whereas Andiracha district showed the lowest (49.58%) mean severity. Logistic regression analysis indicated that EBW incidence of >25% had high probability of association with enset grown on soils with pH of 5.5-7, sole cropped, susceptible clones, using planting materials obtained from other farmers and enset fields with no weeding and EBW management practices. EBW severity of >55% had high probability of association with growing enset in Semen-bench and Yeki districts, weed management through machete slashing, growing local susceptible enset clones, vegetative to maturity growth stages, and low to medium levels of farmer’s awareness about EBW. Findings of this survey indicate that EBW is widely distributed and could be minimized through growing enset preferably on soils out of pH 5.5-7 ranges, intercropping system, proper weeding, access to disease-free planting material, disinfecting farm tools before using, rouging out and burning of infected plants, accessing of advisory services, and limiting free exchange of planting material among enset growers

Multivariate analysis of morphological variation in enset (Ensete ventricosum (Welw.) Cheesman) reveals regional and clinal variation in germplasm from south and south western Ethiopia

Enset (Ensete ventricosum (Welw.) Cheesman) is cultivated by millions of people across Ethiopia in diverse agro-ecological and cultural settings, selecting for various agronomic traits. However, as for other underutilized crops, our understanding of the diversity and utilization of enset remains limited. This work sought to redress this limitation by estimating morphological diversity among enset accessions collected from major enset growing regions, including across altitudinal gradients. In total, landraces comprising 387 accessions originating from nine regions of Ethiopia were characterized using multivariate analysis of 15 quantitative traits. Cluster analysis grouped accessions in to five distinct classes with maximum number of accessions 338 in cluster (I) and minimum 1 in cluster (V). The clustering of accessions did not show grouping on the basis of region of origin. The first four principal components accounted for ~74% of the total variance. Linear discriminant analysis indicated that around 40.8% (160 accessions) and 45.2% (175 accession) of the studied accessions were correctly classified to their respective regions of origin altitude groups, respectively. The breadth of phenotypic differences in these 15 traits suggests significant degrees of genetic variation. These traits will be exploited to identify potential donors for future enset improvement efforts

\u3ci\u3eAspergillus\u3c/i\u3e and aflatoxin in groundnut (\u3ci\u3eArachis hypogaea\u3c/i\u3e L.) and groundnut cake in Eastern Ethiopia

This study was conducted to assess major Aspergillus species and aflatoxins associated with groundnut seeds and cake in Eastern Ethiopia and evaluate growers’ management practices. A total of 160 groundnut seed samples from farmers’ stores and 50 groundnut cake samples from cafe and restaurants were collected. Fungal isolation was done from groundnut seed samples. Aspergillus flavus was the dominant species followed by Aspergillus parasiticus. Aflatoxin analyses of groundnut seed samples were performed using ultra performance liquid chromatography; 22.5% and 41.3% of samples were positive, with total aflatoxin concentrations of 786 and 3135 ng g−1 from 2013/2014 and 2014/2015 samples, respectively. The level of specific aflatoxin concentration varied between 0.1 and 2526 ng g−1 for B2 and B1, respectively. Among contaminated samples of groundnut cake, 68% exhibited aflatoxin concentration below 20 ng g−1, while as high as 158 ng g−1 aflatoxin B1 was recorded. The study confirms high contamination of groundnut products in East Ethiopia

Enset landrace diversity in major enset growing regions of Southern Ethiopia

Enset [Ensete ventricosum (Welw.) Cheesman] is an important food security crop of the Southern Ethiopian highlands. The cultivation of enset is characterised by a wide variety of landraces, suitable to varying agro-ecological conditions and with multiple uses by households. The objective of this paper is to present enset landrace diversity, characteristics and uses in Ethiopia. The study was done through interviews with 375 households covering 20 communities (kebeles) and eight ethnic groups, along an altitudinal range of 1,500 to 3,000 masl across the main enset-producing belt in Southern Ethiopia. A total of 296 locally named enset landraces were recorded. Landrace presence was mostly constrained at the kebele and zone levels, with limited overlap in landrace names across these boundaries. Moderate to high enset landrace diversity was observed on farms across the entire study region. Cultivating a variety of landraces not only allowed for diversified uses, but increases the likelihood of retained yield and food security under variable environmental circumstances. Farmer experience and indigenous knowledge allow for the selection of specific landraces suited to prevalent agro-ecological conditions. We identified a perception bias in the attribution of landrace agro-ecological characteristics, with farmer insight often dependent on the environmental conditions that the local community was exposed to. We underscore the importance of research-based characterisation of enset landraces, to ensure optimal cultivation of this food security crop in changing climatic conditions

Genetic fingerprinting and aflatoxin production of \u3ci\u3eAspergillus\u3c/i\u3e section Flavi associated with groundnut in eastern Ethiopia

Background: Aspergillus species cause aflatoxin contamination in groundnut kernels, being a health threat in agricultural products and leading to commodity rejection by domestic and international markets. Presence of Aspergillus flavus and A. parasiticus colonizing groundnut in eastern Ethiopia, as well as presence of aflatoxins have been reported, though in this region, no genetic studies have been done of these species in relation to their aflatoxin production. Results: In this study, 145 Aspergillus isolates obtained from groundnut kernels in eastern Ethiopia were genetically fingerprinted using 23 Insertion/Deletion (InDel) markers within the aflatoxin-biosynthesis gene cluster (ABC), identifying 133 ABC genotypes. Eighty-four isolates were analyzed by Ultra-Performance Liquid Chromatography (UPLC) for in vitro aflatoxin production. Analysis of genetic distances based on the approximately 85 kb-ABC by Neighbor Joining (NJ), 3D-Principal Coordinate Analysis (3D-PCoA), and Structure software, clustered the isolates into three main groups as a gradient in their aflatoxin production. Group I, contained 98% A. flavus, including L- and non-producers of sclerotia (NPS), producers of B1 and B2 aflatoxins, and most of them collected from the lowland-dry Babile area. Group II was a genetic admixture population of A. flavus (NPS) and A. flavus S morphotype, both low producers of aflatoxins. Group III was primarily represented by A. parasiticus and A. flavus S morphotype isolates both producers of B1, B2 and G1, G2 aflatoxins, and originated from the regions of Darolabu and Gursum. The highest in vitro producer of aflatoxin B1 was A. flavus NPS N1436 (77.98 μg/mL), and the highest producer of aflatoxin G1 was A. parasiticus N1348 (50.33 μg/mL), these isolates were from Gursum and Darolabu, respectively. Conclusions: To the best of our knowledge, this is the first study that combined the use of InDel fingerprinting of the ABC and corresponding aflatoxin production capability to describe the genetic diversity of Aspergillus isolates from groundnut in eastern Ethiopia. Three InDel markers, AFLC04, AFLC08 and AFLC19, accounted for the main assignment of individuals to the three Groups; their loci corresponded to aflC (pksA), hypC, and aflW (moxY) genes, respectively. Despite InDels within the ABC being often associated to loss of aflatoxin production, the vast InDel polymorphism observed in the Aspergillus isolates did not completely impaired their aflatoxin production in vitro

Studier av patosystemet Colletotrichum sublineolum (P. Henn)-Sorgumbicolor (L. Moench) i Etiopia

This thesis covers the assessment of anthracnose incidence and severity in Ethiopia, characterisation of Colletotrichum sublineolum isolates from different sorghum regions of Ethiopia, studying the impact of four selected host genotypes and weather variables on the severity and temporal dynamics of anthracnose, and an evaluation of Ethiopian sorghum accessions for resistance to anthracnose. For these purposes, field surveys were carried out, C. sublineolum isolates were characterised using phenotypic and molecular markers, and two separate field experiments were conducted. The field surveys were conducted in the cropping seasons 2005 and 2007, in 49 districts of six geographic regions. Anthracnose was observed in 41 (84%) of the surveyed districts but both disease incidence and severity varied significantly among the survey districts, geographic regions, altitude groups and climatic zones. Anthracnose incidence ranged from 0 to 77% while severity varied between 0 and 59% across the survey areas. Disease incidence and severity were significantly higher in the lowland (2000 masl). Anthracnose severity was the highest (ca. 40%) in areas characterised by high rainfall (>1200 mm/annum) and intermediate temperature (16-30°C). Areas with moderate rainfall (800-1200 mm/annum) and intermediate or high (>30°C) temperature had a much lower anthracnose severity (<20%). Correlation analysis revealed a significantly positive relationship between rainfall and anthracnose development while temperature did not have a significant effect. In order to study the diversity within C. sublineolum from Ethiopia, sorghum leaves showing anthracnose symptoms were collected from five sampling sites (North, East, South, Southwest 1 and Southwest 2) in four geographic regions, and single spore isolates were categorised into five groups based on their sampling origin. For phenotypic characterisation 50 randomly selected single spore isolates (10 per sampling site) were cultured on PDA plates at 25ºC with four replications. There was significant variation among the isolates in growth rate (1.7-5.8 mm/day, P = 0.0023). However, colony colour and margin showed little variations. For molecular characterisation, amplified fragment length polymorphism analysis (AFLP) was conducted on 102 isolates using six primer combinations. The isolates were highly diverse as revealed by Dice similarity coefficients between individual isolates (0.32-0.96, mean 0.78), average gene diversity for each isolate-group (0.14-0.23, mean 0.19) and proportion of polymorphic peaks per isolate-group (44-68%, mean 58%). Pair-wise genetic distances, and cluster and principal coordinate analyses suggested the genetic separation of the Southern and Eastern isolates from the other groups and also from each other. Overall there was high genetic variation (FST = 0.42) and limited gene flow (Nm = 0.34), and hence such variations should be given due considerations in future sorghum breeding programmes. The impact of four selected host genotypes and weather variables on anthracnose development was studied in a two year field experiment in southern Ethiopia. The four sorghum genotypes (AL70, BTx623, 2001 HararghieColl No. 12 and 2001 PWColl No. 022) were planted in a randomised complete block design with four replications. Initial, final and mean anthracnose severities; and area under the disease progress curves were used as evaluation criteria. Highly significant variations were found among the tested genotypes regardless of the evaluation criteria and experimental year confirming the impact of host genotypes on anthracnose. Anthracnose appeared earlier and progressed rapidly on the susceptible genotype (BTx623) while it appeared late and progressed slowly on the resistant genotype (2001 PWColl No. 022). Initial, final and mean anthracnose severities were all lowest (0.88, 7.56 and 3.57 %, respectively) on 2001 PWColl No. 022 and highest (16.13, 78.38 and 46 %, respectively) on BTx623 across the years. The remaining two genotypes (2001 HararghieColl No. 12 and AL70) had intermediate responses. Correlation and regression analyses revealed a significant impact of rainfall on anthracnose development while temperature appeared to have a non significant effect further confirming the results from the field survey. In another field experiment, a total of 56 Ethiopian sorghum accessions and two susceptible checks (AL70 and BTx623) were evaluated for resistance to anthracnose. Final anthracnose severity (FAS) at 140 days post planting, relative area under the disease progress curve (rAUDPC) and disease progress rate (R) were used as evaluation parameters, and they all varied significantly among the tested accessions. FAS varied between 6.6 and 77.7% while rAUDPC and R ranged from 0.1 to 1.7 and from 0.01 to 0.06 units/day, respectively, over the two years. Analysis of variance revealed significant impact of experimental years, and accession by year interaction on anthracnose development suggesting lack of stable reaction by some accessions. Correlations between the evaluation parameters were high and hence final anthracnose severity was recommended as an efficient and time saving parameter in anthracnose resistance evaluations. All the Ethiopian accessions had significantly lower disease level compared to the susceptible check (BTx623) regardless of the evaluation parameter and experimental year. Fifteen and 18 accessions were rated resistant to anthracnose in 2007 and 2008, respectively, of which six accessions were consistently resistant in both years. The current results suggest the potential Ethiopian sorghum genotypes may have in serving as sources of resistance to anthracnose.Avhandlingen inneholder en kartlegging av forekomst og angrepsgrad av antraknose i Etiopia, en alvorlig sjukdom på sorgum. Isolater fra den patogene soppen Colletotrichum sublineolum samlet i ulike etiopiske sorgumregioner ble karakterisert. Effekter av fire utvalgte sorgum genotyper og klima på antraknose ble studert og resistens mot sjukdommen i etiopiske sorgumlinjer ble bestemt. Soppen ble kartlagt i felt og isolater ble karakterisert ved hjelp av fenotype og molekylære markører. To feltforsøk ble gjennomført. I vekstsesongene 2005 og 2007 ble sjukdommen kartlagt i 49 distrikter lokalisert til seks geografiske regioner. Antraknose ble funnet i 41 (84 %) av de kartlagte distriktene, men det var signifikante forskjeller i både forekomst og angrepsgrad mellom distrikter, geografiske regioner, høydenivåer og klimasoner. Forekomsten av antraknose varierte fra 0 til 77 %, mens angrepsgraden varierte mellom 0 og 59 % i distriktene som ble kartlagt. Forekomsten av antraknose var signifikant høyere i lavlandet (2000 moh). Angrepene av antraknose var sterkest (ca 40 %) i nedbørrike distrikter (>1200 mm årlig) og middels temperatur (16 – 30°C). Distrikter med middels nedbør (800 – 1200 mm) og middels eller høg (>30°C) temperatur hadde mye svakere angrepsgrad (<20%). Det var signifikant positiv korrelasjon mellom nedbør og antraknoseangrep, mens det ikke var signifikant virkning av temperatur. For å studere variasjonen innen C. sublineolum i Etiopia, ble sorgumblad med antraknosesymptomer samlet inn fra fem lokaliteter (nord, øst, sør, sørvest 1 og sørvest 2) i fire geografiske regioner. Ensporeisolater ble delt i fem grupper basert på geografisk opprinnelse. For fenotypisk karakterisering ble 50 tilfeldig valgte ensporeisolater (10 fra hver prøvetatt lokalitet) dyrket på PDA ved 25°C med fire gjentak. Det var signifikante forskjeller i vekstrate mellom isolatene (1.7-5.8 mm/daglig, P=0.0023). Men det var liten forskjell mellom kulturene i farge og veksttype. “Amplified fragment length polymorphism” (AFLP) med seks primerkombinasjoner ble brukt til molekylær karakterisering av 102 isolater. Den store diversiteten mellom isolatene ble vist ved Dice likhetskoeffisient mellom enkelt isolater (0.32-0.96, gjennomsnitt 0.78), gjennomsnittlig gendiversitet for hver isolatgruppe (0.14-0.23, gjennomsnitt 0.19) og andelen polymorfe topper for hver isolatgruppe (44-68%, gjennomsnitt 58%). Parvis genetisk distanse, samt kluster og prinsippiell koordinatanalyse indikerte genetisk separasjon både mellom sørlige og østlige isolater, og mellom disse isolatene og isolater fra andre geografiske områder. Det ble påvist stor genetisk variasjon (FST = 0.42) og begrenset genflyt (Nm = 0.34). Denne variasjonen bør vektlegges i framtidig foredling for resistens mot antraknose. Virkningen av fire sorgum genotyper og klimavariabler på antraknose ble studert i feltforsøk gjennom to vekstsesonger i Sør-Etiopia. De fire genotypene (AL70, BTx623, 2001 HararghieColl No. 12 og 2001 PWColl No. 022) ble plantet i randomiserte, komplette blokkforsøk med fire gjentak. Antraknoseangrepene tidlig og sent i sesongen samt gjennomsnitt ble bestemt. Arealet under kurven for sjukdomsutvikling ble også brukt til vurdering av genotypene. Det var sterk signifikant variasjon mellom genotypene uavhengig av vurderingskriterier og forsøksår. Dette bekreftet betydningen av genotypene for resistens mot antraknose. Sjukdommen startet tidligere og utviklet seg raskere på den mottakelige genotypen (BTx623), mens den kom senere og utviklet seg saktere på den resistente genotypen (2001 PWColl No. 022). Tidlige, sene og gjennomsnitt angrep av antraknose var svakest (henholdsvis 0.88, 7.56 og 3.57%) på 2001 PWColl No. 022 og sterkest (henholdsvis 16.13, 78.38 og 46%) på BTx623 over år. Angrepsgraden på de to andre genotypene 2001 HararghieColl No 12 og AL70) lå mellom de to ytterpunktene. Korrelasjons- og regresjonsanalyser viste signifikant virkning av nedbør på utviklingen av antraknose, mens temperaturen ikke hadde signifikant virkning. Disse resultatene samsvarer med data fra kartleggingen i felt. I et annet feltforsøk ble 56 etiopiske sorgumlinjer og to mottakelige kontroller (AL70 og BTx623) bedømt for resistens mot antraknose. Angrepsgrad ved slutten av sesongen (FAS) 140 dager etter planting, relativt areal under kurven for sjukdomsutvikling (rAUDPC) og fart på sjukdomsutviklingen (R) ble brukt som parameter ved vurderingen. Det var signifikant variasjon mellom linjene for alle disse parametrene. Gjennom to år var FAS mellom 6.6 og 77.7%, rAUDPC var mellom 0.1 og 1.7 og R varierte mellom 0.01 og 0.06 enheter/dag. Variansanalyse viste signifikant virking av forsøksår og samspill mellom år og sorgumlinje. Dette tydet på ustabil reaksjon hos noen linjer. Det var sterk korrelasjonen mellom vurderte parmetre. Derfor kan vurdering ved slutten av vekstsesongen tilrås som effektiv og tidsbesparende evaluering av resistens mot antraknose. Alle etiopiske linjer hadde signifikant svakere angrep av antraknose enn den mottakelige kontrollen (BTx623) ved alle vurderingskriterier og i begge forsøksår. I 2007 ble 15 linjer vurdert som resistente, mens i 2008 ble 18 linjer bedømt som resistente. Seks linjer var resistente begge år. Resultatene viser at etiopiske sorgumlinjer har potensial som kilder for resistens mot antraknose