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This study builds on previous work in which Mask R-CNN was used to quantify common rust severity on maize leaves grown in a greenhouse environment. The Mask R-CNN outperformed standard image processing techniques when applied to the rust quantification task. One of the challenges with using Mask R-CNN is the large amount of parameters that need to be tuned, this study examines automated tuning of these parameters. This study is the first to perform hyper-parameter tuning on the Mask R-CNN at this scale as well as using a genetic algorithm (GA). The results gathered from the GA implementation is compared to that of the prior results from the Mask R-CNN with manually selected hyper-parameters. This work concludes that using a genetic algorithm to determine optimal hyper-parameter values for the Mask R-CNN is comparable to the current state of the art for this problem domain. Future work includes performing a fitness landscape analysis and potential development of new genetic operators.

Cercospora zeina is a causal pathogen of gray leaf spot (GLS) disease of maize in Africa. This fungal pathogen exhibits a high genetic diversity in South Africa. However, little is known about the pathogen’s population structure in the rest of Africa. In this study, we aimed to assess the diversity and gene flow of the pathogen between major maize producing countries in East and Southern Africa (Kenya, Uganda, Zambia, Zimbabwe, and South Africa). A total of 964 single-spore isolates were made from GLS lesions and confirmed as C. zeina using PCR diagnostics. The other causal agent of GLS, Cercospora zeae–maydis, was absent. Genotyping all the C. zeina isolates with 11 microsatellite markers and a mating-type gene diagnostic revealed (i) high genetic diversity with some population structure between the five African countries, (ii) cryptic sexual recombination, (iii) that South Africa and Kenya were the greatest donors of migrants, and (iv) that Zambia had a distinct population. We noted evidence of human-mediated long-distance dispersal, since four haplotypes from one South African site were also present at five sites in Kenya and Uganda. There was no evidence for a single-entry point of the pathogen into Africa. South Africa was the most probable origin of the populations in Kenya, Uganda, and Zimbabwe. Continuous annual maize production in the tropics (Kenya and Uganda) did not result in greater genetic diversity than a single maize season (Southern Africa). Our results will underpin future management of GLS in Africa through effective monitoring of virulent C. zeina strains.

Exserohilum turcicum (sexual stage Setosphaeria turcica) is the hemibiotrophic causal agent of northern leaf blight of maize and sorghum. This study aimed to identify the genes involved in host colonization during the biotrophic and necrotrophic phases of infection. It also aimed to identify race-specific differences in gene expression. RNAseq of maize seedlings inoculated with a race 13N or 23N E. turcicum isolate was conducted before inoculation and at 2, 5, 7, and 13 days post-inoculation (dpi). Biological replicates were pooled per time point for each race and sequenced. A bioinformatics pipeline was used to identify candidate effectors, and expression was validated for selected candidates. Fungal biomass was positively correlated with the percentages of E. turcicum reads mapped, which were low at early time points (2-7 dpi) with a significant increase at 13 dpi, indicating a lifestyle switch from biotrophy to necrotrophy between 7 and 13 dpi. AVRHt1 is the putative E. turcicum effector recognized by the maize resistance gene Ht1. Consistent with this, AVRHt1 was expressed in planta by race 23N, but transcripts were absent in race 13N. In addition, specific transposable elements were expressed in 23N only. Genes encoding the virulence-associated peptidases leupeptin-inhibiting protein 1 and fungalysin were expressed in planta. Transcriptional profiles of genes involved in secondary metabolite synthesis or cell wall degradation revealed the importance of these genes during late stages of infection (13 dpi). A total of 346 expressed candidate effectors were identified, including Ecp6 and proteins similar to the secreted in xylem (SIX) effectors common to formae speciales of Fusarium oxysporum, SIX13 and SIX5. Expression profiling of Ecp6 and SIX13-like indicated a peak in expression at 5 and 7 dpi compared to 2 and 13 dpi. Sequencing of SIX13-like from diverse isolates of E. turcicum revealed host-specific polymorphisms that were mostly non-synonymous, resulting in two groups of SIX13-like proteins that corresponded to the maize or sorghum origin of each isolate. This study suggests putative mechanisms whereby E. turcicum causes disease. Identification of the candidate effector SIX13-like is consistent with the infection mode of E. turcicum through the xylem of susceptible hosts.

Blue light plays an important role in the growth and development of fungi. Environmental cues enable plant pathogenic fungi to synchronise essential metabolic pathways to that of their hosts to gain a competitive advantage. Phylogenetic analysis of the LOV domain present in blue light receptors across all three kingdoms suggests that these receptors in fungal lineages have undergone convergent evolution to use the same domain for control and regulation of similar cellular and metabolic processes. In this review, the genetic basis of blue light photoperception in fungi, and the functions it regulates, will be discussed. Furthermore, the evolution of the light sensing domain and its role in pathogenesis is hypothesised concluding with how knowledge of conserved LOV domains may be exploited for fungal disease control in crop plants.

The genomes of Cercospora zeina, Fusarium pininemorale, Hawksworthiomyces lignivorus, Huntiella decipiens, and Ophiostoma ips are presented in this genome announcement. Three of these genomes are from plant pathogens and otherwise economically important fungal species. Fusarium pininemorale and H. decipiens are not known to cause significant disease but are closely related to species of economic importance. The genome sizes range from 25.99 Mb in the case of O. ips to 4.82 Mb for H. lignivorus. These genomes include the first reports of a genome from the genus Hawksworthiomyces. The availability of these genome data will allow the resolution of longstanding questions regarding the taxonomy of these species. In addition these genome sequences through comparative studies with closely related organisms will increase our understanding of how these species or close relatives cause disease.

Gray leaf spot (GLS) is an important foliar disease of maize. This disease, caused by Cercospora zeina, is prevalent in both smallholder and commercial maize farms in South Africa. Notably, smallholder practices are geared towards conservation agriculture, planting diverse maize genotypes within a field and avoiding chemical control. This study examined the population genetic structure of 129 C. zeina isolates from three smallholder farm sites in KwaZulu-Natal in South Africa using 13 microsatellite markers. These were analysed, together with 239 isolates previously analysed from four commercial farms in the same province, to determine whether farming systems influence the genetic diversity of C.zeina. In addition, we wanted to determine whether the smallholder farming system harboured a greater diversity of C.zeina haplotypes due to lack of chemical spraying of these crops. Overall, farming systems exhibited partial, but significant, population differentiation, contributing 10% of the genetic variation observed. A 16% genetic variation conferred between KwaNxamalala (smallholder) and Cedara (commercial) areas that are in close proximity, confirmed this. Private alleles accounted for 29% of the 52 alleles observed in smallholder farms. Smallholder farms harboured a higher gene and genotypic diversity, with a clonal fraction of only 13% compared to 32% in commercial farms. Mating type ratios indicative of sexual recombination and lower linkage disequilibrium in most smallholder populations were consistent with higher levels of diversity. This study suggests that commercial farming practices, such as fungicides and monoculture crop planting, may result in a narrower genetic diversity of the pathogen that is then propagated by asexual reproduction. In contrast, management of GLS disease in smallholder farms should consider the greater diversity of pathogen genotypes, especially if future research shows that this equates to a greater diversity of pathogenicity alleles.

The genomes of Cercospora zeina, Fusarium pininemorale, Hawksworthiomyces lignivorus, Huntiella decipiens, and Ophiostoma ips are presented in this genome announcement. Three of these genomes are from plant pathogens and otherwise economically important fungal species. Fusarium pininemorale and H. decipiens are not known to cause significant disease but are closely related to species of economic importance. The genome sizes range from 25.99 Mb in the case of O. ips to 4.82 Mb for H. lignivorus. These genomes include the first reports of a genome from the genus Hawksworthiomyces. The availability of these genome data will allow the resolution of longstanding questions regarding the taxonomy of these species. In addition these genome sequences through comparative studies with closely related organisms will increase our understanding of how these species or close relatives cause disease.

The Agropolis Advanced Research Platform, Montpellier, France, the CNRS-South Africa bilateral research agreement and the National Research Foundation, South Africa.

Studies were undertaken to assess the induction of defence response pathways in pearl millet (Pennisetum glaucum) in response to infection with the leaf rust fungus Puccinia substriata. Pretreatment of pearl millet with salicylic acid (SA) conferred resistance to a virulent isolate of the rust fungus, whereas methyl jasmonate (MeJA) did not significantly reduce infection levels. These results suggest that the SA defence pathway is involved in rust resistance. In order to identify pearl millet genes that are specifically regulated in response to SA and not MeJA, and thus could play a role in resistance to P. substriata, gene expression profiling was performed. Substantial overlap in gene expression responses between the treatments was observed, with MeJA and SA treatments exhibiting 17% co‐regulated transcripts. However, 34% of transcripts were differentially expressed in response to SA treatment, but not in response to MeJA treatment. SA‐responsive transcripts represented genes involved in SA metabolism, defence response, signal transduction, protection from oxidative stress and photosynthesis. The expression profiles of pearl millet plants after treatment with SA or MeJA were more similar to one another than to the response during a compatible infection with P. substriata. However, some SA‐responsive genes were repressed during P. substriata infection, indicating possible manipulation of host responses by the pathogen.

The aim of our study was to identify the causal agent of grey leaf spot disease of maize in southern Africa. Single-conidial cultures were recovered from maize leaves with typical disease symptoms sampled from several fields in South Africa, Zambia and Zimbabwe. Morphology, cultural characteristics, and a PCR-based test using Cercospora zeae-maydis and C. zeina-specific primer sets identified all single-conidial cultures as C. zeina. In addition, sequence alignment of DNA fragments of the internal transcribed spacer region (ITS1, ITS2, and the 5.8S gene) and elongation factor 1-α grouped all cultures in the same clade as the C. zeina ex-type culture CBS 118820. To by-pass cultivation of the slow-growing fungus, a rapid method to isolate DNA directly from lesions was successfully applied for PCR identification of C. zeina with species-specific ITS and histone primers. Koch’s postulates were fulfilled for C. zeina by artificially inoculating maize plants in a greenhouse, re-isolating conidia emerging from lesions and verifying pathogen identity with molecular techniques. These results provide evidence that confirms the presence of C. zeina and absence of C. zeae-maydis in commercial maize plantations in southern Africa.

INTRODUCTIONThe identification of differentially expressed genes and examination of their patterns of expression are important to gain information about the functions relevant to processes such as cell differentiation, morpho-logical or metabolic changes, and disease development. Various molec-ular techniques exist for studying dif-ferential gene expression, including representational difference analysis, suppression subtractive hybridiza-tion (SSH), differential display, dif-ferential hybridization, subtractive library construction, serial analysis of gene expression, and cDNA microar-rays (1–6). In previous studies, SSH libraries were screened to identify cloned differentially expressed genes by colony blot hybridization or cDNA amplified fragment length polymor-phism (AFLP) (7,8). However, these methods are time-consuming and do not allow the level of enrichment of a transcript to be quantified. SSH has also been used as a method to gen-erate a cDNA library to use in sub-sequent cDNA microarray expres-sion profiling (9). We used cDNA microarrays to screen PCR-amplified clones from SSH libraries to iden-tify genes from pearl millet [Pennis-etum glaucum (L.) R.Br] and banana (Musa acuminata) that are up-regu-lated during defense responses. This quantitative approach of determining the extent to which transcripts were enriched by the SSH process allowed us to identify and exclude clones that were not derived from up-regulated transcripts and to determine whether transcripts were rare or abundant.MATERIALS AND METHODSPlant Material and Growth ConditionsPearl millet breeding lines ICML12=P7 and 842B were obtained from the International Crops Research Institute for the Semi-Arid Tropics (ICR-ISAT) India and ICRISAT Zimbabwe, respectively. The seed was sterilized and germinated on half-strength Murashige and Skoog (MS) medium (10) at 25°C with a 16 h light/8 h dark photoperiod. Banana plants of a Fusarium oxysporum f.sp. cubense (Foc)-tolerant Cavendish selection and a Foc-susceptible Williams cultivar were transferred into plastic cups containing water and maintained in a greenhouse at 18°–25°C with a 16 h light/8 h dark photoperiod. Prior to inoc-ulation, the plants were transferred to an aeroponic system for root inoculation.Plant TreatmentsLeaves of 10-day-old ICML12=P7 and 842B pearl millet seedlings were wounded by pricking leaves at 1 cm intervals with a sterile needle. The undersurfaces of the leaves were inoc-ulated with a total of 100 μL of either 100 mg/mL chitin or a crude boiled extract of flagellin from Bacillus sp. Control pearl millet 842B seedlings

Background Eucalyptus species and their hybrids encompass approximately 40% of forestry plantation area in South Africa and contribute significantly to the paper pulp industry due to their favourable wood fibre properties. Eucalypt plantation trees are affected by numerous pathogens during their lifetime, some of which can cause severe losses such as Phytophtora spp and Chrysoporthe spp. Plant defence mechanisms against pathogens is currently better understood in the model plant Arabidopsis thaliana where it has been shown that the salicylic acid (SA) and jasmonic acid (JA) signalling pathways enhance resistance against biotrophic and necrotrophic pathogens, respectively [1] . This process involves the up-regulation of specific defence genes which are considered to be marker (diagnostic) genes for the two signalling pathways [2,3].

Grapevine (Vitis vinifera) is the most widely cultivated and economically important fruit crop, but is susceptible to a large number of diseases. Downy mildew, caused by the obligate biotrophic oomycete pathogen Plasmopara viticola, is a common disease present in all regions where vines are cultivated. We used suppression subtractive hybridization (SSH) to generate two cDNA libraries enriched for transcripts induced and repressed, respectively, in the susceptible grapevine cultivar Chasselas 24 h after inoculation with P. viticola. Differential screening on glass slide microarrays yielded over 800 putative genes that were up-regulated in response to P. viticola infection and over 200 that were down-regulated. One hundred and ninety four of these, were sequenced, identified and functionally categorised. Transcript abundance of twelve genes over a 48 h time course was examined by reverse transcriptase quantitative real-time PCR (RT-qPCR). Ten of these genes were induced/enhanced by P. viticola challenge, confirming the results of the SSH. The vast majority of the genes identified are related to defence. Interestingly, many genes involved in photosynthesis were down-regulated.

SUMMARY Fusarium wilt, caused by the fungal pathogen Fusarium oxysporum f. sp. cubense (Foc), is one of the most destructive diseases of bananas. In the tropics and subtropics, Cavendish banana varieties are highly susceptible to Foc race 4 (VCG 0120). Cavendish selection GCTCV-218 was shown to have significantly lower disease severity and incidence compared with susceptible cultivar Williams in replicated greenhouse and field trials. Suppression subtractive hybridization (SSH) was previously carried out to identify genes induced in roots of GCTCV-218, but not in Williams, after infection with Foc'subtropical' race 4. Seventy-nine SSH clones were sequenced and revealed 13 non-redundant gene fragments, several of which showed homology to defence-associated genes, including cell wall-strengthening genes. Quantitative RT-PCR was used to confirm up-regulation and differential expression of a number of genes throughout a time-course, following Foc infection in the tolerant GCTCV-218 when compared with susceptible cv. Williams. Tolerance of GCTCV-218 was linked to significantly increased induction of cell wall-associated phenolic compounds.

A quantitative screening method was developed to evaluate the quality of cDNA libraries constructed by suppression subtraction hybridization (SSH) or other enrichment techniques. The SSH technique was adapted to facilitate screening of the resultant library on a small number of glass slide microarrays. A simple data analysis pipeline named SSHscreen using "linear models for microarray data" (limma) functions in the R computing environment was developed to identify clones in the cDNA libraries that are significantly differentially expressed, and to determine if they were rare or abundant in the original treated sample. This approach facilitates the choice of clones from the cDNA library for further analysis, such as DNA sequencing, Northern blotting, RT-PCR, or detailed expression profiling using a custom cDNA microarray. Furthermore, this strategy is particularly useful for studies of nonmodel organisms for which there is little genome sequence information.

A quantitative screening method was developed to evaluate the quality of cDNA libraries constructed by suppression subtraction hybridization (SSH) or other enrichment techniques. The SSH technique was adapted to facilitate screening of the resultant library on a small number of glass slide microarrays. A simple data analysis pipeline named SSHscreen using "linear models for microarray data" (limma) functions in the R computing environment was developed to identify clones in the cDNA libraries that are significantly differentially expressed, and to determine if they were rare or abundant in the original treated sample. This approach facilitates the choice of clones from the cDNA library for further analysis, such as DNA sequencing, Northern blotting, RT-PCR, or detailed expression profiling using a custom cDNA microarray. Furthermore, this strategy is particularly useful for studies of nonmodel organisms for which there is little genome sequence information.