Sources of resistance to Pseudocercospora fijiensis,the cause of black Sigatoka in banana

Black Sigatoka, caused by Pseudocercospora fijiensis, is one of the most devastating diseases of banana. In commercial banana-growing systems, black Sigatoka is primarily managed by fungicides. This mode of disease management is not feasible for resource-limited smallholder farmers. Therefore, bananas resistant to P. fijiensis provide a practical solution for managing the disease, especially under smallholder farming systems. Most banana and plantain hybrids with resistance to P. fijiensis were developed using few sources of resistance, which include Calcutta 4 and Pisang Lilin. To broaden the pool of resistance sources to P. fijiensis, 95 banana accessions were evaluated under field conditions in Sendusu, Uganda. Eleven accessions were resistant to P. fijiensis. Black Sigatoka symptoms did not progress past Stage 2 (narrow brown streaks) in the diploid accessions Pahang (AA), Pisang KRA (AA), Malaccensis 0074 (AA), Long Tavoy (AA), M.A. Truncata (AA), Tani (BB), and Balbisiana (BB), a response similar to the resistant control Calcutta 4. These accessions are potential sources of P. fijiensis resistance and banana breeding programmes can use them to broaden the genetic base for resistance to P. fijiensis.     

Distribution of Pseudocercospora species causing Sigatoka leaf diseases of banana in Uganda and Tanzania

Sigatoka leaf diseases are a major constraint to banana production. A survey was conducted in Tanzania and Uganda to assess the distribution of Pseudocercospora species and severity of Sigatoka leaf diseases. Pseudocercospora species were identified using species-specific primers. Sigatoka-like leaf diseases were observed in all farms and on all cultivars, but disease severity varied significantly (P < 0.001) between countries, districts/regions within countries, altitudinal ranges and banana cultivars. In all regions except Kilimanjaro, P. fijiensis, the causal agent of black Sigatoka, was the only pathogen associated with Sigatoka disease. Mycosphaerella musae was associated with Sigatoka-like symptoms in Kilimanjaro region. Black Sigatoka disease was more severe in Uganda, with a mean disease severity index (DSI) of 37.5%, than in Tanzania (DSI = 19.9%). In Uganda, black Sigatoka disease was equally severe in Luwero district (mean DSI = 40.4%) and Mbarara district (mean DSI = 37.9%). In Tanzania, black Sigatoka was most severe in Kagera region (mean DSI = 29.2%) and least in Mbeya region (mean DSI = 11.5%). Pseudocercospora fijiensis, the most devastating sigatoka pathogen, was detected at altitudes of up to 1877 m a.s.l. This range expansion of P. fijiensis, previously confined to altitudes lower than 1350 m a.s.l. in East Africa, is of concern, especially for smallholder banana farmers growing the susceptible East African Highland bananas (EAHB). Among the banana varieties sampled, the EAHB, FHIA hybrids and Mchare were the most susceptible. Here, the loss of resistance in Yangambi KM5, a banana variety previously resistant to P. fijiensis, is reported for the first time.     

Pathogenic variation of Mycosphaerella species infecting banana and plantain in Nigeria

Mycosphaerella species that cause the ‘Sigatoka disease complex’ account for significant yield losses in banana and plantain worldwide. Disease surveys were conducted in the humid forest (HF) and derived savanna (DS) agroecological zones from 2004 to 2006 to determine the distribution of the disease and variation among Mycosphaerella species in Nigeria. Disease prevalence and severity were higher in the HF than in the DS zone, but significant (P < 0·001) differences between agroecological zones were only observed for disease severity. A total of 85 isolates of M. fijiensis and 11 isolates of M. eumusae were collected during the survey and used to characterize the pathogenic structure of Mycosphaerella spp. using a putative host differential cultivar set consisting of Calcutta‐4 (resistant), Valery (intermediate) and Agbagba (highly susceptible). Area under disease progress curve (AUDPC) was higher on all cultivars when inoculated with M. eumusae than with M. fijiensis, but significant (P < 0·05) differences between the two species were only observed on Valery. Based on the rank‐sum method, 8·3% of the isolates were classified as highly aggressive and 46·9% were classified as aggressive. About 11·5% of all the isolates were classified as least aggressive, and all of these were M. fijiensis. The majority of M. eumusae isolates (seven out of 11; 64%) were classified as aggressive. A total of nine pathotype clusters were identified using cluster analysis of AUDPC. At least one M. fijiensis isolate was present in all the nine pathotype clusters, while isolates of M. eumusae were present in six of the nine clusters. Isolates in pathotype clusters III and V were the most aggressive, while those in cluster VIII were the least aggressive. Shannon’s index (H) revealed a more diverse Mycosphaerella collection in the DS zone (H = 1·81) than in the HF (H = 1·50) zone, with M. fijiensis being more diverse than M. eumusae. These results describe the current pathotype structure of Mycosphaerella in Nigeria and provide a useful resource that will facilitate screening of newly developed Musa genotypes for resistance against two important leaf spot diseases of banana and plantain.     

Systemic resistance to bacterial leaf speck pathogen in Arabidopsis thaliana induced by the culture filtrate of a plant growth-promoting fungus (PGPF) Phoma sp. GS8-1

The culture filtrate (CF) from the plant growth-promoting fungus Phoma sp. GS8-1 was found to induce systemic resistance in Arabidopsis thaliana against the bacterial leaf speck pathogen Pseudomonas syringae pv. tomato DC3000 (Pst), and the underlying mechanism was studied. Roots of A. thaliana were treated with CF from GS8-1, and plants expressed a clear resistance to subsequent Pst infection; disease severity was reduced, and proliferation of pathogen was suppressed. Various mutants of A. thaliana were used to test whether the CF induced resistance through one of the known signaling pathways: salicylic acid (SA), jasmonic acid (JA), and ethylene (ET). The CF was fully protective against Pst in Arabidopsis mutants jar1 and ein2 similar to wild-type plants. However, its efficacy was reduced in plants containing transgene NahG. Examination of systemic gene expression revealed that CF modulates the expression of SA-inducible PR-1, PR-2 and PR-5 genes, the JA/ET-inducible ChitB gene, and the ET-inducible Hel gene. Moreover, the expression of these genes was further enhanced upon subsequent stimulation after attack by Pst. Our data suggest that in addition to a partial requirement for SA, the signals JA and ET may also play a role in defense signaling in Arabidopsis.     

Differential induction of pathogenesis-related proteins in banana in response to Mycosphaerella fijiensis infection

Black leaf streak or “black Sigatoka” is one of the most important diseases affecting bananas and plantains worldwide. Very few studies have been published on the host-pathogen interaction of this pathosystem, particularly at the molecular level. The aim of this work was to analyze, under controlled conditions, the enzyme activity of peroxidase (POX), phenylalanine ammonia lyase (PAL), β-1, 3-glucanase (GLU) and chitinase (CHI) as well as the production of H₂O₂ in banana plants infected with Mycosphaerella fijiensis. Defence responses were examined and compared in a resistant (Calcutta 4) and a susceptible (Williams) cultivar. Plants were inoculated and tested for enzyme activity at 0, 6, 12, 18, 24, 48 and 72?h after infection (HAI) and 6, 9, 12, 15 and 18?days after inoculation (DAI). A rapid induction of PAL, POX and GLU was observed in the resistant cultivar at 6–18 HAI as well as H₂O₂ production at 72 HAI. In contrast, in the susceptible cultivar, induction of these enzymes was only observed from 6 DAI. These results suggest that the first 72 HAI are important in determining the response of the host to the disease. Further studies characterizing banana responses to M. fijiensis at the early stages of the infection are necessary in order to better understand this host-pathogen interaction.     

Phytoanticipins from banana (<Emphasis Type="Italic">Musa acuminata</Emphasis> cv. Grande Naine) plants,with antifungal activity against <Emphasis Type="Italic">Mycosphaerella fijiensis</Emphasis>, the causal agent of black Sigatoka

The previously detected antifungal activity against Mycosphaerella fijiensis of aqueous infusions of healthy banana (Musa acuminata cv. Grande Naine) leaves, suggested the production of phytoprotectants by the plant. The bioassay-guided VLC-purification of the lyophilized infusion of the leaves of 4-month old healthy banana (M. acuminata cv. Grande Naine) plants, resulted in the purification of a phytoanticipin with strong antifungal activity against M. fijiensis Morelet, the causal agent of black Sigatoka, the most destructive and devastating disease of bananas and plantains in the world. The LC-MS analysis of the purified phytoanticipin suggests a steroidal saponin structure with four sugar units attached to the C-3 position of a diosgenin-like aglycone. This represents the first report of phytoanticipins occurring in M. acuminata.     

Nonspecific toxins as components of a host‐specific culture filtrate from Fusarium oxysporum f. sp. cubense race 1

Bananas and plantains (Musa spp.) are among the most important crops in the world providing staple food for hundreds of millions of people. However, banana production has been devastated by fungal infestations caused by Fusarium oxysporum f. sp. cubense (Foc). Despite the fact that there is very little known on the role of microbial metabolites in the molecular mechanism of Foc infections, it has been proposed that the toxins fusaric acid and beauvericin produced by Foc play an important role during pathogenesis. The aim of this contribution was to study the toxic components of culture filtrates (CF) of Foc and to isolate the extracellular microbial metabolites involved in the plant response. An in vitro bioassay was used to evaluate the production of phytotoxic metabolites as well as the specificity of culture from a strain of Foc belonging to VCG 01210 (race 1). A host‐specific CF was obtained and the phytotoxic compounds characterized as fusaric acid, beauvericin and fumonisin B1. Despite the presence of these nonspecific toxins, a water‐soluble extract from the CF induced protection to the main phytotoxic fraction, measured by lesion area. This hydrophilic fraction induced a fast and strong response of just jasmonic acid (JA)‐dependent defence genes rather than salicylic acid (SA)‐ and ethylene (ET)‐response genes in resistant cultivars. Extracellular proteins isolated from CF of Foc provide an important source for further investigations on the molecular basis of the interaction between Foc and banana.     

Mode of resistance to respiration inhibitors at the cytochrome bc1 enzyme complex of Mycosphaerella fijiensis field isolates

Field isolates of Mycosphaerella fijiensis, causing black Sigatoka of banana, were characterised for their sensitivity to different inhibitors of the cytochrome bc₁ enzyme complex (Qo respiration inhibitors, strobilurin fungicides), using physiological, biochemical and molecular genetic methods. Strobilurin‐resistant isolates exhibited very high resistance factors both in mycelial growth inhibition and NADH consumption assays. Cross‐resistance was observed among all Qo inhibitors, including trifloxystrobin, azoxystrobin, famoxadone, strobilurin B and myxothiazol. However, the Qi and the cytochrome aa₃ inhibitors, antimycin A and potassium cyanide, respectively, were not cross‐resistant to Qo inhibitors. In sensitive but not in resistant isolates, mixtures of Qo inhibitors and SHAM, an inhibitor of the alternative oxidase (AOX), were more active than the components alone, indicating that the alternative pathway is essential in metabolism, but not causal for resistance. In the cell‐free NADH‐consumption assay, the Qo inhibitors affected the sensitive but not the resistant isolates, suggesting that AOX was not active in sub‐mitochondrial particles. In whole cells, however, the AOX has a basic expression level and is probably not inducible by trifloxystrobin. Sequencing of the cytochrome b gene of sensitive and resistant M fijiensis isolates revealed a difference in the nucleotide sequence leading to a single amino acid change from glycine to alanine at position 143 in the resistant isolate. This change is known to occur also in the naturally tolerant basidiomycete Mycena galopoda. It is suggested that the field isolates of M fijiensis can acquire resistance to Qo inhibitors due to a target site alteration with a single base pair change. Resistant isolates do not seem to contain a mixture of mutated and non‐mutated DNA, indicating a complete selection of resistant mitochondria and a maternally donated mode of resistance. © 2000 Society of Chemical Industry     

Progress in understanding Pseudocercospora banana pathogens and the development of resistant Musa germplasm

Banana and plantain (Musa spp.) are important food crops in tropical and subtropical regions of the world where they generate millions of dollars annually to both subsistence farmers and exporters. Since 1902, Pseudocercospora banana pathogens, Pseudocercospora fijiensis, P. musae and P. eumusae, have emerged as major production constraints to banana and plantain. Despite concerted efforts to counter these pathogens, they have continued to negatively impact banana yield. In this review, the economic importance, distribution and the interactions between Pseudocercospora banana pathogens and Musa species are discussed. Interactions are further scrutinized in the light of an emerging climate change scenario and efforts towards the development of resistant banana germplasm are discussed. Finally, some of the opportunities and gaps in knowledge that could be exploited to further understanding of this ubiquitous pathosystem are highlighted.     

Biological control of Pythium,Rhizoctonia and Sclerotinia in lettuce: association of the plant protective activity of the bacterium Paenibacillus alvei K165 with the induction of systemic resistance

The soilborne fungi Sclerotinia sclerotiorum, Rhizoctonia solani and the oomycete Pythium ultimum are among the most destructive pathogens for lettuce production. The application of the biocontrol agent Paenibacillus alvei K165 to the transplant soil plug of lettuce resulted in reduced S. sclerotiorum, R. solani and P. ultimum foliar symptoms and incidence compared to untreated controls, despite the suppressive effect of the pathogens on the rhizosphere population of K165. In vitro, K165 inhibited the growth of S. sclerotiorum and R. solani but not P. ultimum. Furthermore, the expression of the pathogenesis‐related (PR) gene PR1, a marker gene of salicylic acid (SA)‐dependent plant defence, and of the Lipoxygenase (LOX) and Ethylene response factor 1 (ERF1) genes, markers of ethylene/jasmonate (ET/JA)‐dependent plant defence was recorded. K165‐treated plants challenged with P. ultimum showed up‐regulation of PR1, whereas challenge with R. solani resulted in up‐regulation of LOX and ERF1, and challenge with S. sclerotiorum resulted in up‐regulation of PR1, LOX and ERF1. This suggests that K165 triggers the SA‐ and the ET/JA‐mediated induced systemic resistance against P. ultimum and R. solani, respectively, while the simultaneous activation of the SA and ET/JA signalling pathways is proposed for S. sclerotiorum.     

Colonization of <Emphasis Type="Italic">Arabidopsis</Emphasis> roots by <Emphasis Type="Italic">Trichoderma atroviride</Emphasis> promotes growth and enhances systemic disease resistance through jasmonic acid/ethylene and salicylic acid pathways

Trichoderma spp. are common soil fungi used as biocontrol agents due to their capacity to produce antibiotics, induce systemic resistance in plants and parasitize phytopathogenic fungi of major agricultural importance. The present study investigated whether colonization of Arabidopsis thaliana seedlings by Trichoderma atroviride affected plant growth and development. Here it is shown that T. atroviride promotes growth in Arabidopsis. Moreover, T. atroviride produced indole compounds in liquid cultures. These results suggest that indoleacetic acid-related indoles (IAA-related indoles) produced by T. atroviride may have a stimulatory effect on plant growth. In addition, whether colonization of Arabidopsis roots by T. atroviride can induce systemic protection against foliar pathogens was tested. Arabidopsis roots inoculation with T. atroviride provided systemic protection to the leaves inoculated with bacterial and fungal pathogens. To investigate the possible pathway involved in the systemic resistance induced by T. atroviride, the expression profile of salicylic acid, jasmonic acid/ethylene, oxidative burst and camalexin related genes was assessed in Arabidopsis. T. atroviride induced an overlapped expression of defence-related genes of SA and JA/ET pathways, and of the gene involved in the synthesis of the antimicrobial phytoalexin, camalexin, both locally and systemically. This is the first report where colonization of Arabidopsis roots by T. atroviride induces the expression of SA and JA/ET pathways simultaneously to confer resistance against hemibiotrophic and necrotrophic phytopathogens. The beneficial effects induced by the inoculation of Arabidopsis roots with T. atroviride and the induction of the plant defence system suggest a molecular dialogue between these organisms.     

Identification and differentiation of Phyllosticta species causing freckle disease of banana using high resolution melting (HRM) analysis

Freckle disease of banana is caused by three closely related species of Phyllosticta, namely P. musarum, P. maculata and P. cavendishii. In this study, a high resolution melting (HRM) analysis assay was developed and its potential to identify these three fungal species is reported. The assay, which targets the ITS of the nuclear rDNA of the fungal species, generates three distinct melt profiles for the three Phyllosticta species. It is also able to distinguish a combination of up to three co‐infecting species by generating a deviant melt curve. Thirty‐five fungal cultures and infected herbarium leaf specimens, previously characterized using nucleotide sequencing as belonging to one of the three Phyllosticta species, were used for validation of the HRM analysis assay. The normalized curves generated differentiated all samples, with samples from each species correctly identified. The assay was further evaluated against 18 uncharacterized infected leaf specimens from various geographic locations and the results were verified by subsequent nucleotide sequencing. This HRM analysis assay allows rapid identification and differentiation of the three Phyllosticta species using a single primer pair in a one‐step closed‐tube system without labelled fluorescence probes. This novel assay format has potential for simultaneously identifying and differentiating other closely related species of plant pathogens, as well as the classification of infected historic specimens.     

Physiological effects of the hydrophilic phytotoxins produced by <Emphasis Type="Italic">Mycosphaerella fijiensis</Emphasis>, the causal agent of black sigatoka in banana plants

Although Mycosphaerella fijiensis, the causal agent of black sigatoka disease of banana, has been known to produce numerous lipophilic host-selective (HSTs) and nonhost selective phytotoxins (non-HSTs), only recently we have reported that the pathogen also produces hydrophilic phytotoxins. Here we examined the effect of light on the toxicity of the hydrophilic phytotoxins and estimated the electrolyte leakage and H₂O₂ and superoxide generation in detached banana leaves to study their mode of action at the cellular level. Nonhost plant species were also tested to determine whether the toxins are HSTs or non-HSTs. Our results suggest that the hydrophilic phytotoxins are non-HSTs, that their phytotoxicity is not light dependent, and that they may act at the plasma membrane by altering permeability through oxidative damage, by inducing ROS production as part of their mechanism of action.     

The role of photoprotection in defence of two wheat genotypes against Zymoseptoria tritici

This study provides new insights into the role of photoprotection in preformed and induced defence of two wheat genotypes with contrasting phenotypes to infection by Zymoseptoria tritici. We investigated the mechanisms of the photoprotective response during early infection, including nonphotochemical quenching (NPQ), β-carotene-derived xanthophylls, reactive oxygen species, and the phytohormones abscisic acid (ABA), jasmonic acid (JA), and salicylic acid (SA). Furthermore, we quantified the effects of pathogenesis on photosynthesis, stomatal control, and expression of plant defence molecular markers. The photoprotective mechanism of successful defence involved the qI component of NPQ leading to rapid down-regulation of photosystem II quantum yield and chlorophyll a:b, increased biosynthesis of the xanthophyll neoxanthin and ABA, and the expression of chloroplast-specific enzymes to engage in scavenging of O₂^●−. Elevated ABA in the resistant genotype correlated with preformed leaf defence traits including low stomatal density, increased expression of wax biosynthesis, and lignification. Z. tritici exhibited reduced germination and branching on the resistant host genotype and hijacked stomatal control in both genotypes by enhancing stomatal sensitivity to light. Increased biosynthesis of JA and anthocyanins, in contrast to SA, were quantified in the incompatible interaction. Our results indicate that ABA and JA in antagonistic action to SA were associated with defence in the resistant genotype, Cougar, against Z. tritici.     

Antagonistic activity against rice blast disease and elicitation of host-defence response capability of an endophytic Streptomyces albidoflavus OsiLf-2

Rice blast, caused by the fungus Magnaporthe oryzae, can result in notable yield losses in rice production. The objective of this study was to investigate the potential of a rice endophytic isolate, Streptomyces albidoflavus OsiLf-2, on the control of rice blast and the possible mechanisms involved. In vitro assays displayed a variety of antagonistic effects of OsiLf-2 against different physiological races of M. oryzae, with peak mycelial growth inhibition ranging from 74.1% to 83.0%. In vivo tests of OsiLf-2 showed 18.0% and 19.6% reduction in disease index in greenhouse and field conditions, respectively. The stable active metabolites in its cell-free culture filtrate inhibited the mycelial growth, spore germination and appressorial formation of M. oryzae in a dose-dependent manner. They also possessed strong antifungal capacities toward various phytopathogens in vitro. OsiLf-2 secreted multiple antimicrobial compounds, cell wall degradation enzymes, siderophore, plant hormones, and 1-aminocyclopropane-1-carboxylate deaminase, which might function in direct or indirect resistance to M. oryzae. In addition, a variety of defence responses were induced in OsiLf-2-treated rice, including hydrogen peroxide (H₂O₂) accumulation, callose deposition, defence-related enzymes activation, and elevated expression of salicylic acid (SA) and jasmonic acid (JA) pathways genes, which might contribute in resisting pathogen attack. The significant biological control activity and host defence-stimulation ability of OsiLf-2 suggest that this endophytic actinobacterial strain could be a promising candidate in the management of rice blast disease.     

Systemic resistance induced in Arabidopsis thaliana by Trichoderma asperellum SKT-1, a microbial pesticide of seedborne diseases of rice

BACKGROUND: Trichoderma asperellum SKT-1 is a microbial pesticide of seedborne diseases of rice. To investigate the mechanisms of disease suppression in SKT-1, the ability to induce systemic resistance by SKT-1, or its cell-free culture filtrate (CF), was tested using Arabidopsis thaliana Col-0 plants. RESULTS: Both SKT-1 and its CF elicit an induced systemic resistance against the bacterial leaf speck pathogen Pseudomonas syringae pv. tomato DC3000 in Col-0 plants. Involvement of plant hormones in the induced resistance by SKT-1 and CF was assessed using Arabidopsis genotypes such as the jasmonic acid (JA)-resistant mutant jar1, the ethylene (ET)-resistant mutant etr1, the plant impaired in salicylic acid (SA) signalling transgenic NahG and the mutant npr1 impaired in NPR1 activity. In soil experiments using SKT-1, no significant disease suppression effect was observed in NahG transgenic plants or npr1 mutant plants. Expression levels of SA-inducible genes such as PR-1, PR-2 and PR-5 increased substantially in the leaves of Col-0 plants. Expression levels of JA/ET-induced genes such as PDF1.2a, PR-3, PR-4 and AtVsp1 were also induced, but the levels were not as high as for SA-inducible genes. In a hydroponic experiment using CF from SKT-1, all Arabidopsis genotypes showed an induced systemic resistance by CF and increased expression levels of JA/ET- and SA-inducible genes in leaves of CF-treated plants. CONCLUSION: The SA signalling pathway is important in inducing systemic resistance to colonisation by SKT-1, and both SA and JA/ET signalling pathways combine in the signalling of induced resistance by CF. These results indicate that the response of A. thaliana is different from that found in root treatments with barley grain inoculum and CF from SKT-1. Copyright © 2011 Society of Chemical Industry