Variation in Bombyx mori immune response against fungal pathogen Beauveria bassiana with variability in cell wallβ-1,3-glucan

Entomopathogenic fungi are protected by a cell wall with dynamic structure for adapting to various environmental conditions.β-1,3-Glucan recognition proteins activate the innate immune system of insects by recognizing surface molecules of fungi.However,the associations between pathogenicity and the different components of entomopathogenic fungal cell walls remain unclear.Three Beauveria bassiana strains were selected that have significantly differing virulence against Bombyx mori.The molecular mechanisms underlying the immune response in B.mori were investigated using RNA sequencing,which revealed differences in the immune response to different B.bassiana strains at 12 h post-infection.Immunofluorescence assays revealed thatβ-1,3-glucan content had an opposite trend to that of fungal virulence.β-1,3-Glucan injection upregulated BmβGRP4 expression and significantly reduced the virulence of the high-virulence strain but not that of the medium-virulence or low-virulence strains.BmβGRP4 silencing in B.mori with RNA interference resulted in the opposite virulence pattern,indicating that the virulence of B.bassiana was affected by the cell walls’content ofβ-1,3-glucan,which could be recognized by BmβGRP4.Furthermore,interference with the gene CnA(calcineurin catalytic A subunit)involved inβ-1,3-glucan synthesis eliminated differences in virulence between B.bassiana strains.These results indicate that strains of a single species of pathogenic fungi that have differing cell wall components are recognized differently by the innate immune system of B.mori.

Finns-Kingdom RNA Silencing in Plant-Fungal Pathogen Interactions

Fungal pathogens represent a major group of plant invaders that are the causative agents of many notorious plant diseases. Large quantities of RNAs, especially small RNAs involved in gene silencing, have been found to transmit bidirectionally between fungal pathogens and their hosts. Although host-induced gene silencing （HIGS） technology has been developed and applied to protect crops from fungal infections, the mechanisms of RNA transmission, especially small RNAs regulating trans- kingdom RNA silencing in plant immunity, are largely unknown. In this review, we summarize and discuss recent important findings regarding trans-kingdom sRNAs and RNA silencing in plant-fungal pathogen interactions compared with the well-known RNAi mechanisms in plants and fungi. We focus on the interactions between plant and fungal pathogens with broad hosts, represented by the vascular pathogen Verticillium dahliae and non-vascular pathogen Botrytis cinerea, and discuss the known instances of natural RNAi transmission between fungal pathogens and host plants. Given that HIGS has been developed and recently applied in controlling Verticillium wilt diseases, we propose an ideal research system exploiting plant vasculature-Verticillium interaction to further study trans-kingdom RNA silencing.

Nutrition-Associated Processes Govern Fungal Pathogenicity

Fungal pathogens present a major common threat across human communities:they cause disease and death in humans,animals,and global food crops.The development of anti-fungal therapies is hampered primarily by the lack of knowledge about the molecular mechanisms of fungal pathogenicity at the host-pathogen axis.Many studies have shown that nutrition-associated processes such as autophagy and metal homeostasis play essential roles in fungal virulence during systemic infection.During infection,the host immune system often employs nutrition restriction mechanisms to limit microbial proliferation and facilitate the killing and elimination of invading pathogens.Therefore,fast sensing and adaptation to the levels of environmental nutrients are critical if the fungal pathogen is to survive,replicate,and colonize in the host.Fungal cells have evolved numerous precise mechanisms to respond to various levels of nutrient availability.Studies show that fungal cells manipulate the expression of genes involved in nutrient acquisition,autophagosome formation,toxic nutrient detoxification,or cell morphological transition to counter the anti-fungal strategies of the host.This review of the most recent studies of nutrition-related pathways focuses primarily on autophagy,metal homeostasis,and the nutrition-driven morphological switches found in two major human fungal pathogens,Cryptococcus neoformans and Candida albicans.It is a systematic comparison of the functional divergence of nutrition-related genes in the fungal pathogenicity of these evolutionarily distinct but related fungal species.

A secretory phospholipase A2 of a fungal pathogen contributes to lipid droplet homeostasis,assimilation of insect-derived lipids,and repression of host immune responses

Secretory phospholipase A2s(sPLA2s)are found in a wide range of organisms from bacteria to higher plants and animals and are involved in varied and cellular pro-cesses.However,roles of these enzymes in microbial pathogens remain unclear.Here,an sPLA2(BbPLA2)was characterized in the filamentous insect pathogenic fungus,Beau-veria bassiana.BbPLA2was exclusively expressed in insect hemolymph-derived cells(hyphal bodies),and its expression was induced by insect-derived nutrients and lipids,and nutrient starvation.High levels of secretion of BbPLA2 were observed as well as its distri-bution in hyphal body lipid drops(LDs).Overexpression of BbPLA2 increased the ability of B.bassiana to utilize insect-derived nutrients and lipids,and promoted LD accumula-tion,indicating functions for BbPLA2 in mediating LD homeostasis and assimilation of insect-derived lipids.Strains overexpressing BbPLA2 showed moderately increased vir-ulence,including more efficient penetration of the insect cuticle and evasion of host im-mune responses as compared to the wild type strain.In addition,B.bassiana-activated host immune genes were downregulated in the BbPLA2 overexpression strain,but upregulated by infections with a△BbPLA2 strain.These data demonstrate that BbPLA2 contributes to LD homeostasis,assimilation of insect-derived lipids,and repression of host immune responses.

The pathogenic spectrum of fungal keratitis in northwestern China

I am Na An, from the Shaanxi Key Lab of Ophthalmology, Shaanxi Institute of Ophthalmology,Xi＇an City First Hospital, Xi＇an, Shaanxi Province, China. Fungal keratitis is a severe problem in most developing countries.

黑龙江、四川和海南省稻作区水稻穗腐病病原比较分析

【目的】明确中国黑龙江、四川和海南省稻作区水稻穗腐病病原种类及优势致病菌,为水稻穗腐病的精准防控提供参考。【方法】在黑龙江、四川和海南省的13个水稻主要生产区采集水稻穗腐病样品,通过组织分离和单孢分离法,共分离纯化到568个菌株,对各菌株进行形态学鉴定,结合rDNA-ITS序列分析及科赫氏法则验证,明确13个水稻主要生产区穗腐病病原菌种类,分析其优势菌株及致病特征。【结果】黑龙江省水稻穗腐病病原有弯孢菌属、链格孢属和镰孢菌属3类,分别占总病原菌数的1.10%、83.43%和15.47%,链格孢属为该省份的优势菌种。海南省水稻穗腐病病原有弯孢菌属、镰孢菌属、黑孢霉属、毛色二孢属及蠕孢菌属5类,分别占总病原菌数的1.62%、89.47%、0.81%、1.62%和6.48%,镰孢菌属为该省份的优势菌种。四川省水稻穗腐病病原有弯孢菌属、链格孢属、镰孢菌属、黑孢霉属、蠕孢菌属5类,分别占总病原菌数的23.57%、13.47%、22.86%、16.43%和23.57%,无优势菌种。根据分生孢子及菌落形态差异筛选出具有代表性的23个菌株,结合rDNA-ITS序列分析及科赫氏法则验证,明确了水稻穗腐病的致病菌有弯孢菌属的新月弯孢(Curvularialunata),链格孢属的细交链格孢(Alternariatenuissima)、链格孢(A.alternata)、芸薹链格孢(A.brassicae),镰孢菌属的拟轮枝镰孢(Fusariumverticillioides)、新知镰孢(F.andiyazi)、木贼镰孢(F.equiseti)、变红镰孢(F.incarnatum)、厚垣镰孢(F.chlamydosporum),黑孢霉属的稻黑孢(Nigrosporaoryzae)、球黑孢霉(N.sphaerica),毛色二孢属的可可毛色二孢(Lasiodiplodiatheobromae)及蠕孢菌属的嘴突凸脐蠕孢(Exserohilumrostratum)。分别于孕穗期和抽穗扬花期接种穗腐病致病菌,发现无论是粳稻还是籼稻品种,海南省致病菌在水稻抽穗扬花期接种后平均病情指数均高于孕穗期接种的平均病情指数;黑龙江和四川省的致病菌在抽穗扬花期接种的平均病情指数略低于孕穗期接种的平均病情指数。【结论】中国黑龙江、四川和海南省稻作区水稻穗腐病致病菌具有多样性,黑龙江省以链格孢属为优势菌种,海南省以镰孢菌属为优势菌种,四川省优势菌种不明显。

1008例皮肤浅部真菌感染及其病原菌分析

目的分析皮肤浅部真菌感染病种及致病菌种的种类和分布特点。方法收集2021年7月—2022年7月在青岛大学附属医院皮肤科就诊后拟诊断为皮肤真菌感染的患者皮肤标本,共1008例。取镜检阳性的标本接种于沙氏葡萄糖琼脂(SDA)平板或马铃薯葡萄糖琼脂(PDA)平板上进行真菌培养和鉴定,对培养结果为酵母菌属的真菌采用科马嘉(CHROMagar)显色培养基进行分离鉴定,对于无法直接鉴定的菌落采用VITEK ATB自动细菌鉴定和药敏分析系统进行分析鉴定。取镜检阳性或培养为阳性的结果进行统计分析。结果1008例皮肤浅部真菌感染标本中,病种前三位为甲真菌病244例(24.21%)、花斑糠疹184例(18.25%)、足癣182例(18.06%);致病菌种前三位为红色毛癣菌307株(30.46%)、马拉色菌255株(25.30%)、须癣毛癣菌187株(18.55%);甲真菌病女性多于男性(χ^(2)=8.02,P<0.001),股癣男性多于女性(χ^(2)=29.12,P<0.001),差异有统计学意义。致病菌种在性别构成上均无统计学差异(均P>0.05)。结论本地区皮肤浅部真菌感染主要病种为甲真菌病、花斑糠疹和足癣,主要致病菌种为红色毛癣菌、马拉色菌和癣毛癣菌。临床治疗时应加以重视。

Clinical Analysis of 137 Cases of Fungal Keratitis

Objective: To analyze the epidemiology, clinical diagnosis and treatment of fungal keratitis (FK) in hospitalized patients at the First Affiliated Hospital of Yangtze University in recent years. Methods: A retrospective investigation was conducted on the data of 137 cases of FK in our hospital from January 2019 to December 2022. The epidemiological characteristics, identification results of fungal strains, clinical treatment, and prognosis of the patients were analyzed. Results: Among the 137 FK patients, 89 were males and 48 were females, and the ratio of male to female was 1.85:1, The age of onset was the largest number of patients in 50-59 years old and 60-69 years old. The disease occurred most in autumn, winter and summer farming season (from September to December, January, May, June). 72 cases (52.6%) had a clear history of corneal injury, and 43 cases (31.4%) had a history of plant injury. Other risk factors include eye surface diseases, ophthalmic surgery, and wearing corneal contact lenses. The top three pathogens were fusarium (38.7%), aspergillus (23.3%), and alternaria (17.5%). 101 eyes showed improvement or cure after treatment with medication, 9 eyes underwent corneal stromal injection, 11 eyes were covered with conjunctival flap covering or amniotic membrane transplantation, 12 eyes were covered with corneal transplantation, and enucleation of ocular contents was performed in 4 eyes;Visual acuity was improved or maintained in 123 patients (about 89.8%). Conclusions: The incidence of FK in our hospital was mostly middle-aged and elderly men, mostly caused by corneal injury in the process of agricultural labor. The pathogens were mainly fusarium and aspergillus. The preferred treatment was medication, with severe cases requiring combined surgical treatment. Most patients can maintain or improve their vision after treatment.

湖南省猕猴桃叶片主要真菌病害的rDNA-ITS鉴定及序列分析

为了解湖南省猕猴桃叶部真菌病害病原菌的种类和相对多度,在长沙市、邵阳市、常德市、怀化市、张家界市和湘西土家族苗族自治州等地的猕猴桃种植区采集了‘红阳’‘米良1号’‘翠玉’等6个猕猴桃品种的31份感病叶片样本,对病叶上的病原真菌进行分离、筛选和纯化。通过rDNA-ITS鉴定,结合UNITE数据库注释,统计分离出的病原真菌的种类和相对多度,比较了不同地区和不同寄主品种中的致病真菌的种类和相对多度差异。结果表明:31份感病叶片样本中共分离出3门25属75种316株病原真菌,以链格孢属、炭疽菌属和间座壳属为主要群体,分别占比36.71%、18.04%和14.56%;按照地区分布,菌属数量怀化市最多、其次是邵阳市和湘西州;按照猕猴桃品种分布,菌属数量排名前三的依次为‘红阳’‘米良1号’和‘翠玉’。

石榴果实表面真菌的分离鉴定及致病性测定

【目的】研究新疆喀什石榴果实表面真菌的种类,并测试其对石榴果实的致病能力。【方法】分离纯化石榴果实表面真菌,通过形态学和分子生物学对真菌进行鉴定分类,并通过刺伤接种试验测定真菌对石榴果实的致病性,为新疆石榴果实真菌病害的预防与治理提供数据参考。【结果】分离鉴定了7株不同的石榴表面真菌,其中4株可明显致石榴果实腐烂:琉球曲霉(Aspergillus luchuensis)和意大利青霉(Penicillium italicum)对石榴果皮和籽粒均表现出较高的侵染能力,水葫芦链格孢(Alternaria eichhorniae)仅对籽粒果肉具有较高侵染能力,阿氏青霉(Penicillium adametzioide)对石榴果皮和籽粒侵染能力较弱。红色篮状菌(Talaromyes rubrifaciens)、南极枝孢菌(Cladosporium antarcticum)和煤油霉菌(Amorphotheca resinae)未表现出对果皮和籽粒明显的致病能力。【结论】新疆南疆生态条件下石榴果实主要致病菌较其他适生地存在一定区别。

A LAMP-assay-based specific microbiota analysis reveals community dynamics and potential interactions of 13 major soybean root pathogens

Soybean root diseases are associated with numerous fungal and oomycete pathogens;however,the community dynamics and interactions of these pathogens are largely unknown.We performed 13 loop-mediated isothermal amplification(LAMP)assays that targeted specific soybean root pathogens,and traditional isolation assays.A total of 159 samples were collected from three locations in the Huang-Huai-Hai region of China at three soybean growth stages(30,60,and 90 days after planting)in 2016.In LAMP results,we found that pathogen communities differed slightly among locations,but changed dramatically between soybean growth stages.Phytophthora sojae,Rhizoctonia solani,and Fusarium oxysporum were most frequently detected at the early stage,whereas Phomopsis longicolla,Fusarium equiseti,and Fusarium virguliforme were most common in the later stages.Most samples(86%)contained two to six pathogen species.Interestingly,the less detectable species tended to exist in the samples containing more detected species,and some pathogens preferentially co-occurred in diseased tissue,including P.sojae–R.solani–F.oxysporum and F.virguliforme–Calonectria ilicicola,implying potential interactions during infection.The LAMP detection results were confirmed by traditional isolation methods.The isolated strains exhibited different virulence to soybean,further implying a beneficial interaction among some pathogens.

Antibacterial and Antifungal Activity of Organic and Peptidic Extracts of Ecuadorian Endophytic Fungi

Antibacterial and antifungal activities of 32 organic and 14 peptidic extracts obtained from twelve endophytic fungi of the Collection of Endophytes Quito-Catolica were tested against the pathogenic bacteria Staphylococcus aureus, Pseudomonas aeruginosa, Serratia marcescens, Escherichia coli and Salmonella enteritidis, and against the fungal oomycete Pythium ultimum. It was observed that the extracts of Marasmiellus candidus (CEQCA-O1113), Xylaria laevis (CEQCA-O1399), Fusarium solani (CEQCA-O1393), Diaporthe helianthi (CEQCA-O1394) and Xylaria sp. (CEQCA-O1400) partially or totally inhibited the microorganisms tested. The extracts CEQCA-O1399.P1 and CEQCA-O1113.D1 showed fungistatic and fungicidal effects against P. ultimum, respectively. Six extracts strongly inhibited S. aureus, and E. coli was strongly inhibited by the extract CEQCA-O1113.E2. Based on a bioassay-guided approach, the extracts with strong bioactivity in the antibacterial assays by an agar diffusion method were also tested by a disk diffusion method, and the minimal inhibitory concentrations were determined by a microdilution assay. The most bioactive extract, CEQCA-O1113.D1, was also purified by high performance liquid chromatography. The extract recovered its initial bioactivity against S. aureus in two fractions after the purification. Overall, the results of this study highlight the potential of the fungal endophytes as producers of bioactive compounds.

A Study of the Major Pathogens Causing Fruit Rots of Apple in Shelf Life in Hangzhou, Zhejiang Province, China

Major pathogens causing fruit rots of apple in shelf life in Hangzhou, a city in east China, were identified by rDNA-ITS analysis. Their pathogenicities and stress tolerances were compared as well. Combining with disease symptoms, colonial phenotypes and mycelial microscopic morphology, the fungi were determined as Penicillium expansum, Botrytis cinerea, Botryosphaeria dothidea, Diaporthe phaseolorum, Alternaria alternata and Fusarium acuminatum, respectively. Among them, B. cinerea and B. dothidea showed a higher pathogenicity;B. cinerea and D. phaseolorum were hardly affected by the temperature at a range of 15°C and 25°C;B. cinerea has the highest resistant to Thiabendazole and D. phaseolorum displayed the strongest resistance to Imazalil;and P. expansum was most sensitive to ultraviolet light radiation. The results provide some useful information that helps to combine conventional and alternative control strategies to minimize apple postharvest losses in shelf life.

Airborne Fungal Spores of Subalpine Zone of the Karkonosze and Izerskie Mountains(Poland)

The incidence of the airborne fungal spores was determined in the air of subalpine zone of the Karkonosze Mountains and of the Izerskie Mountains in the borderland between Poland and the Czech Republic. The experiment was conducted in2011 and 2012 at three to four week intervals from May to October. Air samples were taken from three locations in the Karkonosze Mts. and one from the Izerskie Mts. To examine the air, the Air Ideal 3P sampler and acidified PDA medium were used. The results show that Cladosporium cladosporioides was the most abundant spore type in all the sampling locations(up to 30%), followed by Alternaria alternata(16%–20%), Fusarium(up to 10%) and Sclerotinia sclerotiorium(up to 6%). The lower spore counts were recorded in May samples, compared to the other months. In this case the snow cover, that was still present in the area at the beginning of May,may be the reason for the lower, compared to June,July and August samplings, CFU(Colony Forming Unit) counts in that month.. The influx of air masses from SE, S and SW sectors in the area under study may affect dissemination of the plant-pathogenic fungi from the Czech Republic and from the South of Europe in general.

2019—2021年四川省血流感染病原真菌分布特征及药敏分析

目的收集全国真菌病监测网四川省中心真菌血流感染数据,整理分析病原菌分布特点及抗真菌药物敏感性情况,为四川省真菌血流感染提供流行病学数据。方法研究28家医院2019年1月1日—2021年12月31日门急诊及住院患者真菌血流感染的数据,采用Whonet 5.6及Microsoft Excle数据透视表功能分析真菌血流感染的临床资料及实验室信息。结果2019年1月1日—2021年12月31日28家医院上报数据中的1220株真菌中,念珠菌属占88.8%(1083/1220),以白念珠菌为主,占35.3%(431/1220);新生隐球菌位于总分离株数的第5位,占8.3%(101/1220)。男性占比56.6%(690/1220),女性患者相比较少(43.4%,530/1220);其中中老年患者(>46岁)79.8%,34.8%来自于重症监护室。白念珠菌对氟康唑的敏感性最高(85.7%);热带念珠菌对氟康唑和伏立康唑的耐药率分别为43.4%和43.3%,且逐年上升。分离的101株新生隐球菌对两性霉素B、氟胞嘧啶、氟康唑、伏立康唑和伊曲康唑均存在不同的非野生株,比例分别为5.0%、2.5%、11.8%、5.7%和8.5%;每家医院分离病原菌的数量排序存在一定差异,大多数医院以白念珠菌为主,但儿童专科性医院近平滑念珠菌分离数最多。结论真菌血流感染病原菌及其抗真菌药物敏感性存在地域性特点,准确掌握所在地区病原菌流行病学资料可以为临床选择抗真菌药物提供依据。

世界卫生组织人类健康中抗微生物药物耐药性的全球优先研究议题

人类健康中的抗微生物药物耐药性全球研究议程确定了40个优先研究主题,旨在为2030年前的政策提供依据。抗微生物药物耐药性对人类健康构成了巨大威胁,据估计,2019年约有495万人的死亡与细菌耐药相关。2021年全球新增耐利福平和耐多药结核病约45万例。此外,全球范围内侵袭性真菌感染正在增加,存在抗真菌药物耐药和诊断困难的挑战。

基于整合方法分析茶树响应病原真菌胁迫的共有模式

为探讨茶树(Camellia sinensis)对病菌胁迫的共有响应模式和抗病机制,运用生物信息学方法对多组RNA-seq数据进行提取、整合及功能富集,结合多种工具和数据库资源对主要调控分子及蛋白互作模块加以分析。结果表明,病原真菌胁迫下,茶树有较多细胞色素P450家族成员表达显著上调;类固醇和激素的代谢过程、苯丙烷合成途径被激活,有丝分裂细胞周期调控、DNA甲基化等生物过程及光合作用途径受到抑制;主要调控分子如转录因子WRKY和NAC、激酶RLK-Pelle和CAMK等以上调为主。差异表达的蛋白互作模块分析表明,有丝分裂周期调控、基于微管运动、淀粉和蔗糖代谢、细胞壁多糖合成、光合作用、类黄酮代谢模块明显下调,木质素合成和萜类生物合成模块上调;且模块之间可能存在互作。病菌胁迫激活的木质素和萜类合成途径的关键基因包括阿魏酸-5-羟基化酶基因F5H、过氧化物酶基因POD和萜类合成酶基因HMGR等。细胞色素P450基因可能在病菌胁迫中起关键作用,增强木质素和萜类物质的合成、削弱光合作用可能是茶树响应真菌胁迫的核心模式。

真菌性角膜炎病原菌分布及药物敏感性结果分析

目的了解我院真菌性角膜炎的病原菌分布情况及其药物敏感性结果,旨在为临床抗真菌药物的选择提供参考。方法采用回顾性病例分析法,收集本院2020年1月至12月角膜炎病变累及部位真菌培养阳性标本86例(来自74例患者),对其病原菌分布和药物敏感性结果进行汇总。结果86例真菌培养阳性标本中,男性患者标本58例,女性患者标本28例,患者的中位年龄为55岁。病变角膜是进行真菌培养的主要取材部位,且培养阳性率高达77.91%。真菌培养阳性率排名前三位的为镰刀菌属、链格孢属和曲霉菌属,且出现1例少见的淡紫紫孢菌。大多数真菌病原菌对伏立康唑敏感。抗真菌药物敏感性结果显示,伏立康唑的敏感率最高,达93.02%;其次为那他霉素,为61.63%;特比奈芬的敏感率为25.58%。氟康唑的耐药率最高,达91.86%;其次为两性霉素,为81.40%;伏立康唑的耐药率最低,为6.98%。结论真菌性角膜炎的最常见病原菌包括镰刀菌属和链格孢属;伏立康唑的药物敏感性最高,其次为那他霉素;氟康唑和两性霉素耐药率较高。在培养结果未出来前,临床医师应经验性使用广谱敏感抗真菌药物,待培养结果出来后应根据药物敏感性结果调整用药。

Botanicals, as a Sustainable Agroecological Alternative to Synthetic Pesticide for Controlling Leaf Miner (Pinworm) and Fusarium Wilt Disease of Tomato

Tomato (Solanum lycopersicum Mill) is a staple fruit vegetable widely grown worldwide for its rich nutrients including vitamins, minerals and proteins. In spite of its importance, yield and fruit quality in Buea, Cameroon is quite low compared to other countries. Pests and diseases are responsible for these limitations. Those of high importance are pinworm (Tatu absoluta) and Fusarium wilt respectively. Synthetic chemicals have been used to manage this pest/disease on tomato but this has not been very successful because of high cost and unavailability of these chemicals. Botanicals are known to have anti-microbial properties. 10% Leeks and marigold extracts were applied to tomato plants one week after planting. Five treatments were replicated four times in a randomized complete block. They include a control, a synthetic insecticide, leeks extract, marigold extract, and leeks/marigold extracts. Growth and yield parameters were evaluated and data obtained was analysed by descriptive statistics and ANOVA. Results showed that more tomato plant leaves were recorded in the leeks + marigold treatment (54) which differed significantly (P = 0.05) from the control (45). More fruits were harvested in the leeks + marigold treatment (32) which differed significantly (P = 0.05) from the control (18). Pinworm numbers were significantly higher in control (6) (P = 0.05) than the other treatments. Numerous leaf damage was observed in control (20) which differed significantly (P = 0.05) across treatments. More fruit damage was seen in control (14) and differed significantly (P = 0.05) from the other treatments. The least disease incidence was noted in Leeks + Marigold treatment (27.5%), and the most in control (72.5%) which differed significantly (P = 0.05) across treatments. Fusarium oxysporium and Fusarium semitectum were identified. There was a significant difference in pathogen number in control compared to other treatments. Thus leeks and marigold extract increased yield of tomato by mitigating the effects of leaf miner and fusarium wilt.