Keke Cheng & Shixiao Yu, from Sun Yat-sen University, discuss their article: Specificity determinants of pathogens in forest in English and Chinese.
Background
Autumn has come and the fruit is ripe. Seeds fall from the tree and spread around the mother tree, attracting squirrels who love to eat them. Squirrels came and ate a large number of seeds and went away satisfied. The few seeds that survive settle in the soil and then in the spring, they begin to take root and grow into seedlings. Unfortunately, the seedlings that sprout around the mother tree are attacked by specialized pathogens or herbivorous insects, so few survive. At the same time, this creates space for the offspring of other species. Over time, this delicate balance has enabled many species to coexist in the forest.
Schematic diagram of the Janzen-Connell hypothesis (left) and the study site located in Heishiding Nature Reserve, Guangdong Province, China (right). Photo by Keke Cheng.
The Janzen-Connell hypothesis was proposed by ecologists in order to reveal the formation and maintenance of biodiversity in forests. It suggests that distance- and density-dependent effects from specialized natural enemies promote species coexistence. Unfortunately, isolating and identifying pathogens and determining their specificity has been challenging because of their small size and difficulties in culturing them. Even when cultured, it is difficult to isolate and identify host-specific pathogens and to describe their specificity to hosts, because of their small and intricate morphology. As a result, decades of research have focused on seedling death caused by fungal communities, rather than revealing the mechanisms of infection by a single pathogen to different hosts. Additionally, observing pathogen specificity in forest communities is challenging because of the complexity of plant-microbe interactions. In fact, it is difficult to observe pathogen specificity in forest communities because of the complexity of plant-microbe interactions, not only the negative effects of pathogens on plants, but also the positive effects of mycorrhizal fungi on plants. This complexity highlights the importance of isolating and culturing pathogens to determine their specificity at the individual fungal level.
Our Study
For our experiment, we collected 113 seedlings from four common tree species in a subtropical forest in southern China. Root fungi were isolated and cultured after disinfection of the root surface of seedlings, and 87 strains of fungi were identified successfully. Then, according to the number of available seedlings, 27 strains of fungi were selected and inoculated back into their own host seedlings for pathogenicity determination. The results showed that six of the 27 strains of fungi had obvious pathogenicity to their own host seedlings. Subsequently, in order to test the host specificity of these pathogens, these six pathogens were inoculated into host plants and four other non-host species. Five of the pathogens, except for Aspergillus niger, caused 30-50% mortality in host seedlings, with no significant effect on other non-host plants. This indicates that these five pathogens have strong host specificity.
Finally, in order to directly demonstrate how host-specific pathogens infect different tree species, we successfully labeled four of these pathogens with green fluorescent protein (GFP). Three of the pathogens were host-specific and the other was the broad-spectrum fungus, A. niger. This labeling method allowed us to track their pathogenic differences in the four tree species. The results clearly show that although all the pathogens are capable of diffusing and growing on the root surfaces of both host and non-host species, the hyphae of each host-specific pathogen can only grow into the vascular tissue of its specific host. This suggests that the pathogenesis of these host-specific pathogens depends on the infection of the vascular system of the host.
Significance of the Study
Unlike previous studies of plant-pathogen interactions, we did not use general sterilized and non-sterilized soil treatments, but isolated and cultured pathogens and labeled them with GFP to directly trace their accumulation in different host tissues. This approach provides a straightforward and intuitive visual way to assess the host specificity of pathogens while revealing their underlying pathogenic mechanisms. While it is still necessary to isolate and culture potential pathogens to determine pathogenicity and specificity, this GFP technique enables direct and comparable visualization of infections in different hosts without harming the plants.
Overall, we visually demonstrated the differences in hyphal growth in vascular tissue of host and non-host plants during infections by host-specific pathogens. Our study lays the foundation for our research into whether host-specific pathogens are able to infect phylogenetically related species and to analyze their potential infection mechanisms. More importantly, the application of GFP technology will help deepen our understanding of the role of pathogens in species coexistence and biodiversity maintenance in forest communities.
借助绿色荧光蛋白技术揭示森林中宿主专一性病原菌的秘密
程珂珂 & 余世孝, 来自中山大学，讨论他们的文章: Specificity determinants of pathogens in forest
背景
秋天来了，果实成熟了。一粒粒种子从树上掉落，散布在母树周围，这吸引来了爱吃它的松鼠。松鼠大快朵颐地吃掉了大量的种子，心满意足地走了。幸存下来的些许种子沉淀在土壤中，随后在春天来临的时候，开始生根发芽长成幼苗。不幸的是，这些萌发在母树周围的幼苗被具有偏好的病原菌或植食性昆虫所觊觎，因此能够存活下来的幼苗寥寥无几。但这却为其他异种后代提供了生存空间。长久以来， 这种微妙的平衡使得在森林维持了多物种的共同生存。
图 1. Janzen-Connell假说示意图（左）和位于中国广东省黑石顶自然保护区的研究地点（右）
生态学家为了揭示森林中生物多样性的形成和维持机制提出了Janzen-Connell 假说。该假说认为，专一性的自然天敌所引起的距离和密度制约能够促进物种共存。这一观点受到越来越多的验证支持。由于病原菌形态小、不易培养等特征，使得分离和鉴定宿主专一性病原菌以及描述病原菌对宿主的特异性程度非常困难。因此，几十年来的研究一直集中在真菌群落导致的幼苗死亡，而不是揭示单一病原菌对不同宿主的感染机制。另外，事实上在森林群落中很难直接观察到病原菌的专一性，因为植物与微生物间的相互作用非常复杂，不仅有病原菌对植物的负作用，还有菌根真菌对植物的正作用。而这就使得分离和培养病原菌以在单个真菌水平上确定其专一性，显得至关重要。
我们的研究
我们从中国南方亚热带森林的四种常见树种中采集了113株幼苗。在对幼苗根部表面进行消毒后分离并培养了根系真菌，并成功鉴定出87株真菌。接着根据可用幼苗的数量，选择了其中27株真菌将其接种回自身的宿主幼苗中进行致病性测定。结果发现27株真菌中有六种真菌对其自身宿主幼苗表现出明显的致病性。随后，为了测试这些病原菌的宿主专一性，将这六种病原菌接种到宿主植物和另外四种非宿主物种中，发现除Aspergillus niger 以外的五株病原菌导致宿主幼苗30%–50%的死亡率，而对其他非宿主植物没有显著影响，这表明这五种病原菌具有较强的宿主专一性。
最后，为了直接揭示宿主专一性病原菌如何侵染不同植物，我们用绿色荧光蛋白技术GFP 成功标记了四种病原菌，其中三种是宿主专一性的，另一种是广谱真菌黑曲霉(A. niger)。这种标记方法使我们能够定位它们在四种树种中的致病差异。结果清楚地表明，尽管所有病原菌都能够在宿主和非宿主植物的根系表面扩散生长，但每种宿主专一性病原菌的菌丝仅只能向其特定宿主的维管组织内生长。这表明这些宿主专一性病原菌的致病机制需要侵染宿主的维管系统。
研究意义
与以往关于植物-病原菌相互作用的研究不同，我们没有采用一般的灭菌和非灭菌土壤处理方法，而是分离和培养病原菌并借助绿色荧光蛋白对其进行标记，以直接追踪其在不同宿主组织中的积累。这种方法提供了一种相对简单且直观可视化的方法来评估病原菌的宿主专一性，同时揭示了潜在的致病机制。虽然仍然需要分离和培养潜在的病原菌来确定致病性和专一性，但这种荧光标记技术可以避免对宿主植物的破坏，并直接且可比较地观察其在不同宿主中的感染过程。
总的来说，我们通过可视化手段直观地展示了宿主专一性病原菌在感染宿主和非宿主植物时，菌丝在维管组织中的生长差异。我们的研究为探讨宿主专一性病原菌是否能够感染系统发育相关的物种以及分析其潜在的感染机制奠定了基础。意义更深刻的是，GFP技术的应用将有助于加深我们了解病原菌在物种共存和森林群落生物多样性维持中作用。
